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PubMed2003

In situ hybridization and immunohistochemistry of versican, aggrecan and link protein, and histochemistry of hyaluronan in the developing mouse limb bud cartilage.

We investigated the expression pattern of versican, aggrecan, link protein and hyaluronan in the developing limb bud cartilage of the fetal mouse using in situ hybridization and/or immunohistochemistry. Versican mRNA and immunostaining were detected in the mesenchymal cell condensation of the future digital bone at E13. Versican mRNA expression rapidly disappeared from the tibial cartilage, as cartilage formation progressed during E13-15, but the immunostaining was gradually replaced by aggrecan immunostaining from the diaphysis. Immunostaining for both molecules thus had a 'nega-posi' pattern and consequently versican immunostaining was still detected at the epiphyseal end at E15. This result indicated that versican functions as a temporary framework in newly formed cartilage matrix. An aggrecan-positive region within the cartilage invariably had intense hyaluronan staining, whereas a versican-positive region also had affinity for hyaluronan within the cartilage, but not in the mesenchymal cell condensation. Therefore, the presence of versican aggregates was not confirmed in the developing limb bud cartilage. Furthermore, although link protein was more closely related with aggrecan than versican during limb bud cartilage formation, there was a discrepancy between the expression of aggrecan and link protein in tibial cartilage at E15. In particular, only a link protein-positive region was present in the marginal area of the metaphysis and the epiphysis at this stage. This finding may indicate a novel role for link protein.

Aggrecans↗
PubMed1998

Correlation between biochemical composition and magnetic resonance appearance of articular cartilage.

OBJECTIVE: The objective of this study was to find a correlation between magnetic resonance (MR) appearance and biochemical composition of the normal articular cartilage by comparing the laminar aspects with the distribution of the two principal matrix components: proteoglycans and collagen. DESIGN: T2-weighted MR microimages of porcine cartilage-bone plugs, excised from both the habitually loaded and habitually unloaded regions of the proximal end of the humerus, were obtained using a spin-echo sequence. Proteoglycans (PGs) were monitored by histology and by measuring the uronate and the sulfur content of the tissue; a histologic method and the chemical determination of hydroxyproline were used for the evaluation of the collagen content. RESULTS: The 'loaded' cartilage exhibited the expected MR laminar appearance whereas the 'unloaded' tissue appeared to be more homogeneous. The PG content in the 'loaded' cartilage, was found to be 2.4 times higher than in the habitually unloaded tissue, exhibiting an increasing trend from the articular surface to the bone. In the 'unloaded' cartilage the uronate distribution was more uniform with a higher concentration in the intermediate zone. The mean collagen content of both cartilage regions was found to be about 39% of the tissue dry weight. Histology and hydroxyproline distribution pattern showed that collagen was particularly concentrated at the surface and in a central zone of the 'loaded' cartilage whereas in the 'unloaded' tissue collagen was evident only at the surface. In accordance with the collagen distribution, transverse relaxation (T2) times in 'loaded' cartilage showed a minimum value at the articular surface and another minimum in a central region. On the contrary, the average T2 value of the 'unloaded' tissue was high at the surface and decreased rapidly in the deeper zones. CONCLUSION: These results demonstrate that the MR appearance of articular cartilage correlates with the collagen content, but not with that of PGs, of the different zones. Other matrix components might, however, influence the MR appearance by contributing to the macromolecular organization of the tissue.

Animals↗
PubMed1998

Collagen type I antisense and collagen type IIA messenger RNA is expressed in adult murine articular cartilage.

OBJECTIVE: Articular cartilage has only limited capacities for repair and it is not known what is the exact mechanism of matrix restoration. It was investigated whether the reparative process in murine articular cartilage after moderate proteoglycan depletion is accompanied by a change in the chondrocyte phenotype either to hypertrophy or to a less differentiated phenotype as assayed by the expression of specific collagen subtypes. DESIGN: Moderate proteoglycan depletion was induced by injection of papain whereafter the expression of collagen type I mRNA, collagen IIA and IIB mRNA and type X collagen mRNA in patellar cartilage, as markers for chondrocyte phenotype, was investigated by RT-PCR during normal cartilage physiology and matrix restoration. In addition, in-situ expression of collagen subtypes was assayed by immunolocalisation. RESULTS: In normal articular cartilage collagen I, collagen IIB and collagen type X transcripts were easily detected. Surprisingly, collagen type I sense as well as antisense mRNA was detected and in addition to IIB transcripts collagen IIA transcripts were detected in a number of samples. During cartilage matrix restoration no change in the expression of collagen I, collagen IIA or IIB or collagen type X mRNA transcripts could be detected. Immunolocalization demonstrated the presence of type I (pericellular) and type II collagen in the extracellular matrix. The pericellular matrix of hypertrophic chondrocytes showed collagen type X staining in the calcified cartilage in normal and papain-injected knee joints. Increased staining for collagen type X was found in the upper cartilage layer in the interterritorial matrix from day 7 after papain injection. CONCLUSION: The absence of changes in collagen mRNA expression indicates that alteration of chondrocyte phenotype does not occur during the successful repair process after moderate proteoglycan depletion. Collagen type X appears to be deposited in the upper cartilage layer during this process.

Animals↗
PubMed2000

Heterogenous response of knee cartilage to pregnancy in the rabbit: assessment of specific mRNA levels.

OBJECTIVE: The purpose of this study was to evaluate the effect of pregnancy on mRNA levels for several relevant molecules between five articular cartilage surfaces of the rabbit knee joint. DESIGN: Total RNA was extracted from the following five knee joint articular surfaces: the lateral and medial femoral condyles (LFC and MFC); the lateral and medial tibial plateau (LTP and MTP); and the femoral groove (G) from pregnant and age-matched non-pregnant skeletally immature New Zealand White rabbits. The RNA was analysed by the sensitive molecular technique of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using rabbit specific primer sets. Two types of comparisons were performed: (i) comparison of mRNA levels between cartilage surfaces and (ii) comparison of mRNA levels between pregnant and non-pregnant rabbits within the same articular surfaces. RESULTS: (i) Total RNA yield from the MFC and G represented 53 and 58% of the total RNA amount from the five cartilage surfaces in both non-pregnant and pregnant rabbits, respectively. Transcript levels for progesterone receptor (PR), aggrecan and biglycan were similar in all of the cartilage surfaces. In contrast, the cartilage surfaces exhibited significantly different transcript levels with a similar pattern for the estrogen receptor (ER), collagenase and urokinase (i.e., MTP<LTP<MFC=G<LFC). In addition, mRNA levels were significantly different for type II collagen (COL 2) between the LFC and MTP, for decorin between the LTP and both LFC and G, for TIMP-1 between the MFC and both the LTP and the G, as well as for PAI-1 between the LTP and MTP. (ii) Total RNA concentration from pregnant animals were significantly decreased in the MFC and the G. For all molecules studied except PR, there was a general tendency for decreased mRNA levels during pregnancy. Compared to non-pregnant rabbits, mRNA levels were significantly decreased in pregnant rabbits for ER in the LFC, LTP and MTP; for COL2 in both LTP and MTP; for aggrecan in the MTP, for biglycan in both LTP and MTP, for decorin in the MTP and G; for collagenase in the LFC, LTP, MTP and G, for urokinase in the LFC, MFC and LTP; for TIMP-1 in the MFC, LTP and MTP. In contrast, PAI-1 exhibited increased mRNA levels during pregnancy in the LFC and LTP. (iii) Comparison of results between the present and the previous study demonstrates that subtle changes in one or more of the cartilage surfaces may be obscured if cartilage is pooled. CONCLUSION: The present study demonstrates that regulation of RNA levels in articular cartilage during pregnancy is complex and variable between cartilage surfaces.

Animals↗
PubMed2000

Validation of high-resolution water-excitation magnetic resonance imaging for quantitative assessment of thin cartilage layers.

OBJECTIVE: To employ a magnetic resonance (MR) imaging technique for quantitative assessment of thin cartilage layers, and to validate the cartilage volume and thickness measurements. METHODS: We investigated 10 normal elbow joints (age 20 to 69 years) with a 3D gradient echo sequence with selective water excitation (TR 18 ms; TE 9 ms; FA 25 degrees, resolution 1x0.25x0.25 mm2, imaging time 19 min). After interpolating the image data to a 0.125x0.125 mm2 in-plane resolution, the cartilage plates were segmented, reconstructed in 3D, and the cartilage volume and thickness determined with a 3D Euclidean distance transformation algorithm, independent of the original section plane. The cartilage volume and thickness values were compared with CT arthrography and A-mode ultrasound. RESULTS: The mean systematic difference between the elbow cartilage volume obtained from MR imaging and CT arthrography was -0.11% (-6.0 mm3) and the mean random difference 5.7% (314 mm3). Except for the fovea capitis radii, the deviations were not statistically significant (range -7.6 to +11.7%). In the humerus, the mean cartilage thickness (average = 1.35 mm) was overestimated relative to CT arthrography (+20.7%/+0.23 mm), and slightly underestimated relative to A-mode ultrasound (-6.0%/-0.05 mm). With few exceptions, there were no significant differences between MRI, CT arthrography and ultrasound in the other joint surfaces of the elbow (random deviations between 0.08 and 0.39 mm). CONCLUSIONS: The technique presented can be applied for determining the cartilage volume and 3D thickness in joints with thin cartilage layers with a reasonable degree of accuracy.

Adult↗
PubMed2000

Tumor necrosis factor alpha can contribute to focal loss of cartilage in osteoarthritis.

UNLABELLED: OBJECTIVE; To evaluate the potential for tumor necrosis factor alpha (TNFalpha)-induced focal loss of cartilage in osteoarthritic (OA) knee joints. DESIGN: Fresh cartilage from specified regions of OA joints was immunostained for TNF-receptor (R) bearing chondrocytes. Cartilage explants from the same regions were cultured with or without small amounts of TNFalpha and cumulative GAG release into supernatants measured. Concentrations of TNFalpha, p55 and p75 soluble (s) TNF-R in supernatants from cultured OA and non-arthritic (NA) synovium were measured by ELISA. RESULTS: TNF-R bearing chondrocytes were identified in OA cartilage; more specimens contained p55 TNF-R- than p75 TNF-R-bearing chondrocytes and differences in TNF-R distribution were apparent in cartilage from different regions of the same knees. TNFalpha at 5, 1, 0.5 and 0.25 ng/ml (but not 0.1 ng/ml) significantly increased glycosaminoglycans (GAG) release from cartilage explants in a dose-dependent manner. Variation in susceptibility to TNFalpha was observed in explants from different sites. TNFalpha and p75 sTNF-R, but not p55 sTNF-R, concentrations were significantly higher in OA, as compared with NA, supernatants. A significant correlation between TNFalpha and p75 sTNF-R measurements was apparent only in NA supernatants. CONCLUSIONS: Variations in chondrocyte TNF-R expression occur in OA cartilage in vivo. TNFalpha at concentrations produced by OA synovium in vitro, can degrade cartilage matrix. In most OA supernatants sTNF-R concentrations were insufficient to abrogate the effects of TNFalpha. Thus conditions exist in some OA knees for TNFalpha to contribute to focal loss of cartilage.

Aged↗
PubMed2000

The effects of glucosamine derivatives on equine articular cartilage degradation in explant culture.

OBJECTIVE: To determine whether glucosamine-3-sulfate, glucose-3-sulfate (control) and N-acetyl glucosamine inhibit experimentally induced degradation of equine articular cartilage explants similar to glucosamine HCl. DESIGN: Articular cartilage was obtained from the antebrachio-carpal and middle joints of horses (2-8 years old) killed for reasons unrelated to lameness. Cartilage discs were harvested from the weight-bearing region of the articular surface and cultured. Media were exchanged daily and the recovered media stored at 4 degrees C. On days 1 and 2 lipopolysaccharide (LPS, 10 microg/ml) was added to induce cartilage degradation. To evaluate the effects of different sources of glucosamine (on an equal molar basis), varying concentrations of glucosamine HCl (0.25, 2.5, or 25 mg/ml), glucosamine-3-sulfate (0.304, 3.04, or 30.4 mg/ml), or N-acetyl-glucosamine (0.256, 2.56, or 25.6 mg/ml) were added to the cultures. The glucose-3-sulfate control was added at 0.3075, 3.075 or 30.75 mg/ml. Nitric oxide and proteoglycan released into conditioned media and tissue proteoglycan synthesis and total tissue PG content were measured as indicators of cartilage metabolism. RESULTS: Glucosamine-3-sulfate consistently inhibited cartilage degradation in a manner similar to glucosamine HCl, while the effects of N-acetyl-glucosamine were highly variable and did not inhibit cartilage degradation. Glucose-3-sulfate did not inhibit cartilage degradation. CONCLUSION: Our results indicate that glucosamine sulfate also has the potential to prevent or reduce articular cartilage degradation similar to glucosamine HCl in vitro. The amine group at the carbon-2 position appears important for the effectiveness of the glucosamine derivative. The therapeutic value of N-acetyl-glucosamine remains questionable.

Acetylglucosamine↗
PubMed2001

Altered mechanics and histomorphometry of canine tibial cartilage following joint immobilization.

Joint immobilization is associated with altered cartilage biosynthesis and catabolism that may affect cartilage mechanics and joint function. In this study, the mechanical behavior of articular cartilage was studied in an experimental model of joint immobilization, in which the canine knee was cast-immobilized at 90 degrees of flexion for 4 weeks. Articular cartilage from the medial tibial plateau was tested in compression and in shear. Biochemical assays for water and glycosaminoglycan content and histomorphometric grading were performed on site-matched samples. Significant decreases in the equilibrium and dynamic shear moduli, but not compressive moduli, were observed in cartilage after 4 weeks of joint immobilization as compared to cartilage from a separate control population. Importantly, there was also evidence of a decrease in the compressive and shear moduli of tibial cartilage from the contralateral knee joints compared to control joints that were not immobilized. No significant effect of immobilization on the biochemical parameters or histomorphometric scores was detected, expect for a significant loss of proteoglycan staining following immobilization. These findings for changes in the tibial cartilage following cast immobilization are consistent with a mild form of cartilage degeneration.

Analysis of Variance↗
PubMed2001

Role of growth factors in rabbit articular cartilage repair by chondrocytes in agarose.

OBJECTIVE: Novel approaches to intervention in joint diseases consist of the replacement of diseased cartilage by in vitro engineered, viable cells or graft tissues. Two major obstacles remain to be overcome: (1) Hyaline cartilage in vitro often loses differentiated traits. (2) Grafts frequently are not integrated satisfactorily into host cartilage and/or the tissue is remodelled in situ into functionally inferior fibrocartilage. Therefore, we have explored the possibility whether chondrocytes embedded into agarose gels provided better graft tissues in a repair model of full thickness defects in rabbit joint cartilage. DESIGN: Experimental defects of knee joint cartilage was filled with articular chondrocytes cultured in agarose gels. Chondrocytes in vitro either remained unstimulated or were treated with several growth factors. Repair of the defects was assessed by histology and was scored between 0 (no healing) and 1 (perfect healing) as judged by the follwing parameters: intensity of proteoglycan staining, organization of the superficial zone, ossification at the border between repair cartilage and subchondral bone, tidemark formation in the repaired area, arrangement of chondrocytes, and integration of repair cartilage into host. RESULTS: Treatment of chondrocyte cultures with bFGF had a stabilizing effect on the differentiated state of the cells in implanted grafts whereas bone morphogenetic proteins stimulated ingrowth of subchondral bone reducing repair cartilage thickness and preventing normal tide mark formation; TGF-beta did not significantly affect evaluation parameters in comparison with untreated controls. CONCLUSION: Growth factor treatment resulted in an ambiguous quality of graft development. Only FGF had a clear beneficial effect to the graft tissues after 1 month. Further studies are required to define the precise conditions and sequence of growth factor treatment of in vitro engineered cartilage which benefits graft quality.

Animals↗
PubMed2001

Contacts with fibrils containing collagen I, but not collagens II, IX, and XI, can destabilize the cartilage phenotype of chondrocytes.

OBJECTIVE: Cell-matrix interactions are important regulators of cellular functions, including matrix synthesis, proliferation and differentiation. This is well exemplified by the characteristically labile phenotype of chondrocytes that is lost in monolayer culture but is stabilized in suspension under appropriate conditions. We were interested in the role of collagen suprastructures in maintaining or destabilizing the cartilage phenotype of chondrocytes. DESIGN: Primary sternal chondrocytes from 17-day-old chick embryos were cultured in gels of fibrils reconstituted from soluble collagen I from various sources. The culture media either contained or lacked FBS. Cells were cultured for up to 28 days and the evolution of the phenotype of the cells was assessed by their collagen expression (collagens II and X for differentiated chondrocytes and hypertrophic chodrocytes, repectively; collagen I for phenotypically modulated cells), or by their secretion of alkaline phosphatase (hypertrophic cartilage phenotype). RESULTS: The cells often retained their differentiated phenotype only if cultured with serum. Under serum-free conditions, cartilage characteristics were lost. The cells acquired a fibroblast-like shape and, later, synthesized collagen I instead of cartilage collagens. Shape changes were influenced by beta1-integrin-activity, whereas other matrix receptors were important for alterations of collagen patterns. Heterotypic fibrils reconstituted from collagens II, IX, and XI did not provoke this phenotypic instability. CONCLUSIONS: Chondrocytes sensitively recognize the suprastructures of collagen fibrils in their environment. Cellular interactions with fibrils with appropriate molecular organizations, such as that in cartilage fibrils, result in the maintenance of the differentiated cartilage phenotype. However, other suprastructures, e.g. in reconstituted fibrils mainly containing collagen I, lead to cell-matrix interactions incompatible with the cartilage phenotype. The maintenance of the differentiated traits of chondrocytes is pivotal for the normal function of, e.g., articular cartilage. If pathologically altered matrix suprastructures lead to a dysregulation of collagen production also in vivo compromised cartilage functions inevitably will be propagated further.

Alkaline Phosphatase↗
PubMed2002

Integrative articular cartilage repair: dependence on developmental stage and collagen metabolism.

OBJECTIVES: The objectives of this research were to determine whether the integrative repair of bovine cartilage explants was dependent on developmental stage, and whether observed differences in integration with developmental stage were related to deposition of newly synthesized collagen and lysyl oxidase-mediated collagen cross-linking. METHODS: Pairs of fetal, newborn calf, and adult bovine cartilage blocks were cultured in partial apposition for 2 weeks in medium supplemented with serum, ascorbate, and [3H]proline. Following culture, mechanical integration between apposed cartilage blocks was assessed by measuring adhesive strength in a single-lap shear configuration. Formation and stabilization of newly synthesized protein and collagen was investigated by determination of [3H]proline and [3H]hydroxyproline in tissue digests and guanidine extracts. RESULTS: Calf cartilage exhibited a relatively high integrative repair phenotype, achieving an adhesive strength that was three--four-fold that of adult or fetal specimens. The low and high integrative repair phenotypes appeared related in part to different levels of collagen biosynthesis, which was approximately four--five-fold higher in calf cartilage samples than in the adult. However, fetal cartilage also exhibited a high level of biosynthesis. The different integrative repair phenotypes were not associated with marked differences in the kinetics of chemical stabilization of newly synthesized collagen, as the proportion of incorporated [3H]proline and newly-formed [3H]hydroxyproline that was resistant to extraction by 4M guanidine-HCl following culture was similar for cartilage from all developmental stages. Integration of calf cartilage appeared to depend on lysyl oxidase-mediated collagen cross-link formation, since inclusion of beta-aminopropionitrile (BAPN) in the culture medium completely eliminated development of adhesive strength. BAPN treatment also increased the percentage of newly synthesized protein in the guanidine extracts from 10% to 36% of the total, and that of newly synthesized collagen from 2% to 20%, while having only slight inhibitory effects on overall protein and collagen biosynthesis. CONCLUSION: The finding that cartilage exhibits enhanced integrative repair at a certain developmental stage suggests that it may ultimately be possible to enhance repair when needed in clinical situations.

Animals↗
PubMed2002

The effects of immobilization on the characteristics of articular cartilage: current concepts and future directions.

OBJECTIVE: The purpose of this paper is to review current data and concepts concerning the effect of immobilization on articular cartilage in animal models. We also evaluate the methods to measure articular cartilage changes in humans. METHODS: Studies looking at the effects of immobilization on morphological, biochemical, and biomechanical characteristics of articular cartilage are reviewed. RESULTS: Articular cartilage changes in immobilized animals include altered proteoglycan synthesis, as well as thinning and softening of the tissue. The overall thickness of articular cartilage in the knee decreases up to 9% after 11 weeks of immobilization and the deformation rate under test load increases up to 42%. Quantitative data about changes in human articular cartilage following immobilization are not available. This is mainly due to the lack of an accurate, reproducible, and non-invasive method to characterize articular cartilage. DISCUSSION: An understanding of the alterations in articular cartilage following short and long term immobilization in humans is essential for the optimization of rehabilitation programs. Refined imaging techniques combined with state-of-the-art visualization tools could allow the systematical monitoring of articular cartilage morphology changes in immobilized humans.

Animals↗
PubMed2002

Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen and Hoechst 33258.

OBJECTIVE: To compare two fluorometric assays, utilizing (1) the bisbenzimidazole Hoechst 33258 and (2) PicoGreen, for determining DNA content in human cartilage. METHODS: Human articular and nasal septal cartilage explants were digested using proteinase K. Portions of sample digest were analysed for intrinsic and dye-enhanced fluorescence with either Hoechst 33258 or PicoGreen. RESULTS: Intrinsic tissue fluorescence in both articular and septal cartilage increased with age and was prominent at wavelengths used for Hoechst 33258 but relatively low at wavelengths used for PicoGreen. The relative contribution of intrinsic fluorescence to total dye-enhanced fluorescence of human cartilage was markedly greater for Hoechst 33258 (19-57%) than for PicoGreen (2-7%). Thus, in many situations, DNA in human cartilage can be assayed using PicoGreen without the need to correct for intrinsic cartilage fluorescence. The enhancement of fluorescence by each dye was found to be specific for DNA, as shown by fluorescence spectra, >90% sensitivity to DNase, and resistance to RNase. In addition, little or no interference was caused by non-DNA tissue components, since DNA caused an equal enhancement in the absence or presence of proteinase K digested human cartilage, once intrinsic cartilage fluorescence was subtracted. PicoGreen was more sensitive for assaying DNA (0.9ng DNA/ml) than Hoechst 33258 (6ng DNA/ml) and can also be used in a microplate reader. CONCLUSION: PicoGreen can be used in a rapid and sensitive assay to quantify DNA in small samples of human cartilage.

Adult↗
PubMed2000

[Antibiotic impregnation of cartilage implants: diffusion kinetics of fluoroquinolones].

BACKGROUND: Antibiotic impregnation of cartilage implants may reduce the risk of bacterial infection and subsequent absorption. The aim of this study was to investigate the penetration kinetics of two quinolone antibiotics into fresh cartilage and the concentrations in the core of lyophilized cartilage after rehydration. METHODS: Fresh human costal cartilage was impregnated with ofloxacin and ciprofloxacin (2 mg/ml) for 2, 15 and 90 min. Concentrations were measured in 6 levels (0.5 mm each) from the surface to 3 mm beneath the surface with high performance liquid chromatography (HPLC). Lyophilized human costal cartilage was rehydrated in ofloxacine and ciprofloxacine solutions (2 mg/ml, 0.2 mg/ml and 0.02 mg/ml) for 18 hours and concentrations in the core of the rib segment were measured. RESULTS: Quinolone antibiotics penetrate into cartilage by free diffusion. We found no evidence of significant binding to cartilage. After 2 and 15 min of impregnation, concentrations above the minimal inhibitory concentration (MIC90) for pseudomonas species are found from 0-0.5 mm below the surface of fresh cartilage. After 90 min concentrations above the MIC90 were found 1.0-1.5 mm below surface of the implant. In lyophilized rib grafts which were rehydrated in 1/10 diluted intravenous solutions (0.2 mg/ml), concentrations in the core of the specimen were above MIC90. Differences between the penetration characteristics of ofloxacine and ciprofloxacine were minor. CONCLUSION: Intraoperative impregnation of cartilage implants with ofloxacin or ciprofloxacin probably offers only short-term protection against bacterial infection. Rehydrated rib grafts, however, contain high quinolone concentrations which may be effective even in infected implant beds for several hours.

Anti-Infective Agents↗
PubMed1997

[Transplantation of in vitro cultured cartilage materials: characterization of matrix synthesis].

BACKGROUND: Recently a three-dimensional model for the formation of cartilage in vitro was developed. The aim of this study was to investigate the amount and quality of newly synthesized matrix after graftig in vitro engineered cartilage into athymic nude mice. MATERIAL AND METHODS: Group I received transplants consisting of human chondrocytes, agarose, and E 200 (a bioabsorbable polymer fleece that offers mechanical stability. Ethicon Inc). Group II received chondrocytes and agarose only. At intervals of six, 12, and 24 weeks after subcutaneous transplantation we used azan blue staining and antibodies against collagen type I, collagen type II, and chondroitin-4sulfate to characterize the matrix synthesis. A quantitative analysis was performed using the computer image analyzing software photoshop (Adobe Inc). RESULTS: In group I, the amounts of newly synthesized cartilage specific collagen type II and chondroitin-4 sulfate increased progressively. Twenty-four weeks after transplantation, these amounts were comparable to the original human cartilage from which the chondrocytes were derived. Collagen type I was detected only in small quantities in the periphery of the transplants. Gross examination revealed sufficient mechanical stability and unremarkable changes in size and form. In contrast to this, group II transplants showed markedly smaller amounts of cartilage specific matrix components as collagen type II and chondroitin-4 sulfate and at the same time greater amounts of collagen type I. It was found both in the periphery and in central parts of the transplants. There was a remarkable loss of volume in all transplants and mechanical stability was poor. CONCLUSIONS: The absorbable cell carrier E 200 not only offers mechanical stability to in vitro engineered cartilage but also had a positive effect on the development of cartilage in our experiments. In conclusion, in vitro engineered cartilage is a promising pathway for the replacement of cartilage defects.

Adolescent↗
PubMed1986

Effect of physical exercise on indentation stiffness of articular cartilage in the canine knee.

Using the indentation method, we investigated the effects of physical exercise (treadmill running 4 km/day for 15 weeks) on stiffness of the articular cartilage in the canine knee. Considering cartilage to be an elastic material with homogeneity and isotropy, we calculated elastic moduli for femoral, tibial, and patellar cartilages using instant and 15-s deformations after load application. Although the elastic moduli do not represent, because of the non-equilibrium condition, true elastic properties of articular cartilage, they characterize, together with the retardation time spectrum, the integrated response of articular cartilage during the first 15 s after load application. The moderate loading used in our training program caused no macroscopic changes on the articular surface. In running dogs, the mean stiffness of articular cartilage increased by 6% as compared with the controls. Stiffening, which was attributed to the decreased fluid flow in the cartilage, was significant (P less than 0.05) on the patellar surface of the femur and on the tibial condyles. In general, stiffness increased more (approximately 10%) in the cartilage areas, which were repeatedly heavily loaded during running exercise. This alteration in the biomechanical property as well as an increase (approximately 11%) in thickness were considered to be typical responses of articular cartilage to an increased, but physiologic loading pattern.

Animals↗
PubMed1997

Identification of chondromodulin I as a novel endothelial cell growth inhibitor. Purification and its localization in the avascular zone of epiphyseal cartilage.

Cartilage is unique among tissues of mesenchymal origin in that it is resistant to vascular invasion due to an intrinsic angiogenic inhibitor. During endochondral bone formation, however, calcified cartilage formed in the center of the cartilaginous bone rudiment allows vascular invasion, which initiates the replacement of cartilage by bone. The transition of cartilage from the angioresistant to the angiogenic status thus plays a key role in bone formation. However, the molecular basis of this phenotypic transition of cartilage has been obscure. We report here purification of an endothelial cell growth inhibitor from a guanidine extract of bovine epiphyseal cartilage. The N-terminal amino acid sequence indicated that the inhibitor was identical to chondromodulin I (ChM-I), a cartilage-specific growth-modulating factor. Purified ChM-I inhibited DNA synthesis and proliferation of vascular endothelial cells as well as tube morphogenesis in vitro. Expression of ChM-I cDNA in COS7 cells indicated that mature ChM-I molecules were secreted from the cells after post-translational modifications and cleavage from the transmembrane precursor at the predicted processing signal. Recombinant ChM-I stimulated DNA synthesis and proteoglycan synthesis of cultured growth plate chondrocytes, but inhibited tube morphogenesis of endothelial cells. In situ hybridization and immunohistochemical studies indicated that ChM-I is specifically expressed in the avascular zone of cartilage in developing bone, but not present in calcifying cartilage. These results suggest a regulatory role of ChM-I in vascular invasion during endochondral bone formation.

Animals↗
PubMed2000

Reduced NO accumulation in arthrotic cartilage by exposure to methylene blue.

Nitric oxide (NO) appears to be a final common inflammation mediator of cartilage degradation. Halting the pathological formation of excessive NO, by suppressing the inducible NO synthase (iNOS) activity, may help to preserve cartilage integrity. We used fresh ex-vivo human articular cartilage explants from normal and arthrotic joints for assessment of NO levels, as determined by its nitrite degradation products and nitric oxide synthase expression. We measured matrix proteoglycan content, assessed by image analysis of alcian blue staining, and proteoglycan synthesis, assessed by sulfate incorporation into proteoglycans. The effect of methylene blue, a nitric oxide synthase inhibitor, on matrix preservation was evaluated. Cartilage discs in vitro, derived from normal appearing joints, secreted about one tenth as much NO compared to discs derived from arthrotic cartilage. Cartilage explants showed a time-dependent reduction in the amount of aggrecan within the cartilaginous matrix. Addition of methylene blue to the growth medium lowered nitric oxide accumulation and prevented matrix degradation in the cultured cartilage discs. The cartilage matrix preservation effect was mediated through downregulation of all three isoforms of NOS, i.e., the neuronal NOS, endothelial NOS and inducible NOS and upregulation of TGF beta receptor in the chondrocytes. Our findings indicate that inhibition of NOS activity preserves cartilage matrix in vitro.

Cartilage, Articular↗