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Diffusion of small solutes in cartilage as measured by nuclear magnetic resonance (NMR) spectroscopy and imaging.

The ability of water and solutes to move through the cartilage matrix is important to the normal function of cartilage and is presumed to be altered in degenerative diseases of cartilage such as osteoarthritis and rheumatoid arthritis. Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) techniques were used to measure a self diffusion coefficient (D) for small solutes in samples of explanted cartilage for diffusion times ranging from 13 ms to 2 s. With a diffusion time of 13 ms, the intratissue diffusivity of several small solutes (water, Na+, Li+, and CF3CO2-) was found consistently to be about 60% of the diffusivity of the same species in free solution. Equilibration of the samples at low pH (which titrates the charge groups so that the net matrix charge of -300 mM at pH 8 becomes approximately -50 mM at pH 2) did not affect the diffusivity of water or Na+. These data, and the similarity between the D in cartilage relative to free solution for water, anions, and cations, are consistent with the view that charge is not an important determinant of the intratissue diffusivity of small solutes in cartilage. With 35% compression, the diffusivity of water and Li+ dropped by 19 and 39%, respectively. In contrast, the diffusivity of water increased by 20% after treatment with trypsin (to remove the proteoglycans and noncollagenous proteins). These data and the lack of an effect of charge on diffusivity are consistent with D being dependent on the composition and density of the solid tissue matrix. A series of diffusion-weighted proton images demonstrated that D could be measured on a localized basis and that changes in D associated with an enzymatically depleted matrix could be clearly observed. Finally, evidence of restriction to diffusion within the tissue was found with studies in which D was measured as a function of diffusion time. The measured D for water in cartilage decreased with diffusion times ranging from 25 ms to 2 s, at which point the measured D was roughly 40% of the diffusivity in free solution. Although changes in matrix density by compression or digestion with trypsin led to a decrease or increase, respectively, in the measured D, the functional change in measured diffusivity with diffusion time remained essentially unchanged. In a different type of study, in which bulk transport could be observed over long periods of time, cartilage was submerged in 99% D2O and MRI studies were performed to demonstrate the bulk movement of water out of the cartilage matrix.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Electric field stimulation can increase protein synthesis in articular cartilage explants.

It has been hypothesized that the electric fields associated with the dynamic loading of cartilage may affect its growth, remodeling, and biosynthesis. While the application of exogenous fields has been shown to modulate cartilage biosynthesis, it is not known what range of field magnitudes and frequencies can alter biosynthesis and how they relate to the magnitudes and frequencies of endogenous fields. Such information is necessary to understand and identify mechanisms by which fields may act on cartilage metabolism. In this study, incorporation of 35S-methionine was used as a marker for electric field-induced changes in chondrocyte protein synthesis in disks of cartilage from the femoropatellar groove of 1 to 2-week-old calves. The cartilage was stimulated sinusoidally at 1, 10, 100, 10(3), and 10(4) Hz with current densities of 10-30 mA/cm2. Incorporation was assessed in control disks maintained in the absence of applied current at 37, 41, and 43 degrees C. The possibility that applied currents would induce synthesis of the same stress proteins that are caused by heating or other mechanisms was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examination of gel fluorographs. Total radiolabel incorporation in cartilage that had been stimulated relative to incorporation in the controls increased with current density magnitudes greater than 10 mA/cm2. The increase was greatest at 100 Hz and 1 kHz, and it depended on the position on the joint surface from which the cartilage samples were taken. Together, these results suggest that endogenous electric fields could affect cartilage biosynthesis. Stress proteins were not induced at any current density when the electrodes were electrically connected but chemically isolated from the media by agarose bridges. Stress proteins were observed for disks incubated at temperatures greater than 39 degrees C (no field) and when the stimulating platinum electrodes were in direct contact with the media bathing the cartilage disks. Therefore, the increase in incorporation of 35S-methionine due to applied fields with the use of chemically isolated electrodes did not appear to be associated with stress response.

Animals↗

Quantitation of articular cartilage using magnetic resonance imaging and three-dimensional reconstruction.

A quadrature knee coil was used in conjunction with a magnetic resonance imaging scanner for quantitation of test phantom volumes, ex vivo bovine cartilage thickness, and in vivo human articular cartilage volumes. Optimal magnetic resonance parameters were obtained by testing a series of spin-echo and gradient-echo pulse sequences to determine the sequence that provided the highest resolution of articular cartilage and best defined the cartilage interfaces with synovial fluid and subchondral bone. Extensive testing revealed that two sequences were required to define articular cartilage accurately: a spoiled gradient-echo sequence and a steady state free-precession sequence. Three-dimensional reconstruction and statistical analyses of test phantoms and of bovine and human cartilage images were performed. Differences between actual phantom volumes and three-dimensional measurements demonstrated that, as magnetic resonance slice thickness was increased, the measurement variability also increased (coefficient of variation ranging from 1.7 +/- 1.3% for 1.0 mm slice thickness to 22.7 +/- 1.9% for 3.0 mm slice thickness). When the phantom volume was greater than 1,800 mm3, the intraobserver, interobserver and interscan accuracies were greater than 97, 98, and 96%, respectively. This high degree of reproducibility pertained for the data on in vivo human cartilage data also. For experienced observers, the intraobserver and interobserver reproducibility were greater than 98 and 97%, respectively. The interscan reproducibility was greater than 98%. These data demonstrate that improved magnetic resonance pulse sequencing, in conjunction with three-dimensional reconstruction and measurement techniques, can accurately and reproducibly measure the volume of articular cartilage. Clinical application of this approach offers the potential for early diagnosis of osteoarthritis and for serial, noninvasive assessment of changes in articular cartilage volume in response to therapeutic modalities.

Adult↗

Depth-dependent confined compression modulus of full-thickness bovine articular cartilage.

The objective of this study was to determine the equilibrium confined compression modulus of bovine articular cartilage as it varies with depth from the articular surface. Osteochondral samples were compressed by 8, 16, 24, and 32% of the cartilage thickness and allowed to equilibrate. Intratissue displacement within the cartilage was measured with use of fluorescently labeled chondrocyte nuclei as intrinsic, fiducial markers. Axial strain was then calculated in nine sequential 125 microns thick cartilage layers comprising the superficial 1,125 microns and in a 250 microns thick layer of cartilage adjacent to the cartilage-bone interface. Adjacent osteochondral cores were also tested in confined compression to determine the equilibrium stresses required to achieve the same levels of compression. Stress-strain data for each layer of each sample were fit to a finite deformation stress-strain relation to determine the equilibrium confined compression modulus in each tissue layer. The compressive modulus increased significantly with depth from the articular surface and ranged from 0.079 +/- 0.039 MPa in the superficial layer to 1.14 +/- 0.44 MPa in the ninth layer. The deepest layer 250 microns thick, had a modulus of 2.10 +/- 2.69 MPa. These moduli were markedly different from the apparent "homogeneous" modulus for full-thickness cartilage (0.38 +/- 0.12 MPa) and ranged from 21 to 560% of that value. The relatively low moduli and the compression-induced stiffening of the superficial layers suggest that these layers greatly affect the biomechanical behavior of cartilage, such as during confined compression testing. The delineation of the depth-dependent modulus provides a basis for detailed study of the relationship between the composition, structure, and function of cartilage in such processes as aging, repair, and degeneration.

Animals↗

Cultured human ankle and knee cartilage differ in susceptibility to damage mediated by fibronectin fragments.

According to numerous cadaveric, radiographic, and clinical studies, ankle and knee joints differ in susceptibility to osteoarthritis. To test for biochemical differences in susceptibility to damage, a chondrocytic chondrolysis system has been utilized. In this system, fibronectin fragments are added to cultured cartilage explants, resulting in enhanced release of catabolic cytokines, induction of matrix metalloproteinases, temporary suppression of proteoglycan synthesis, and consequently, severe loss of cartilage proteoglycan. We found that the addition of an amino-terminal thrombin-generated 29-kDa fibronectin fragment to cultured knee cartilage from 14 donors (average age: 53 years) usually caused a 30-50% decrease in proteoglycan content by day 7. However, of the ankle cartilage specimens examined from 21 donors (average age: 50 years), only three showed damage by day 7, one by day 14, and six by day 21, and 11 were not damaged until day 28. For eight of the donors (average age: 44 years), both knee and ankle cartilages were obtained: this allowed comparison between tissues from the same donor. The analysis showed that the ankle cartilage was much more refractory to damage than was the knee cartilage from the same donor. These data clearly show differences between ankle and knee cartilage in susceptibility to the fibronectin fragments and suggest the feasibility of use of these fragments for discerning differences in homeostasis of the ankle and knee cartilage.

Adolescent↗

Deformation of articular cartilage collagen structure under static and cyclic loading.

Relatively little is known about the morphology of articular cartilage under conditions of normal use, yet a more profound knowledge is both critical to the understanding of cartilage function and helpful for the validation of tissue-engineered cartilage. In this study, the deformation of the articular cartilage of the tibial plateau under compressive static and cyclic loading is characterized. Whole knee joints of rabbits were loaded ex vivo while the knee was held statically or allowed to move against resistance. Load magnitudes of quadriceps were maintained at either three (high) or one (low) times body weight for 30 minutes. For cyclic loading, the tibia was flexed between 70 and 150 degrees relative to the femur at 1 Hz with either a cyclic or constant force. The recovery of cartilage after unloading was examined for each loading condition. At the end of the loading, specimens were cryofixed while under load, freeze-substituted, and prepared for scanning electron microscopy. Morphological examination demonstrated significantly higher deformation of the collagen structure throughout all cartilage zones under static loading conditions compared with cyclic loading conditions in which deformation was limited to the superficial regions. The minimum thickness of the cartilage that remained after loading was dependent on the magnitude of load and was significantly smaller with static loads (54% of the thickness of the unloaded controls) than after cyclic loading or constant-force cyclic loading (78 or 66% of the thickness of the unloaded controls, p < 0.05). Acute bending of the collagen fibers was observed under both loading conditions: in the superficial half of the articular cartilage after static loading and in the superficial quarter after cyclic loading. Complete recovery of all deformation occurred within 30 minutes but was significantly faster after cyclic loading. These data suggest that the structure of the collagen of articular cartilage exhibits a zone-specific deformation that is dependent on the magnitude and type of load.

Animals↗

Integrative cartilage repair: inhibition by beta-aminopropionitrile.

The effects of beta-aminopropionitrile, a known inhibitor of lysyl oxidase, on the extractability of newly synthesized collagen and integrative cartilage repair were determined in explant cultures of adult bovine articular cartilage. Dose-escalation studies indicated that treatment of cartilage explants for 6 days with beta-aminopropionitrile caused a dose-dependent inhibition of proteoglycan synthesis ([35S]sulfate incorporation) with a 50% inhibition at 2.2 mM. However, 0.25 mM beta-aminopropionitrile had no detectable effect on proteoglycan synthesis and was thus used for subsequent experiments. Treatment of cartilage with beta-aminopropionitrile for 14 days increased the extractability of newly synthesized collagen with 4 M guanidine-HCl while having little effect on proteoglycan synthesis, proteoglycan deposition, collagen synthesis (formation of [3H]hydroxyproline after labeling with [3H]proline), collagen deposition, or cartilage cellularity (DNA content). In untreated cultures, the percentage of radiolabeled collagen ([3H]hydroxyproline) that was extractable after 1 day of radiolabeling, 6 days of radiolabeling, or 6 days of label and 6 days of chase decreased from 81 to 25 and 9%, respectively. In beta-aminopropionitrile-treated cultures, the extractability was relatively higher (96, 62, and 47%, respectively). Treatment with beta-aminopropionitrile after radiolabeling with [14C]lysine also significantly inhibited the formation of the reducible crosslink [14C]dihydroxylysinonorleucine without affecting the overall deposition in cartilage of [14C]lysine and [14C]hydroxylysine. In functional repair studies, treatment with beta-aminopropionitrile caused an almost complete inhibition of integration between pairs of cartilage explants maintained in apposition for 2 weeks. These results indicate that beta-aminopropionitrile blocks the formation of collagen crosslinks in cartilage explants and suggest that such crosslinks are critical to integrative cartilage repair.

Aminopropionitrile↗

Comparison of biomechanical and biochemical properties of cartilage from human knee and ankle pairs.

Cartilage was obtained from eight matched knee (tibiofemoral and femoropatellar) and ankle (talocrural) joints of five different donors (both left and right from donors 14, 22, and 38 years of age, and left only from donors 31 and 45 years of age) within 24 hours of death. All cartilage was graded as normal by the macroscopic visual Collins' scale and the histological Mankin scale. Cylindrical disks of cartilage were harvested from 10 sites within the tibiofemoral and femoropatellar joint surfaces and four sites within the talocrural joint, and uniaxial confined compression measurements were performed to quantify a spectrum of physical properties including the equilibrium modulus, hydraulic permeability, dynamic stiffness, streaming potential, electrokinetic coupling coefficient, and electrical conductivity. Matched specimens from the same 14 sites were used for complementary measurements of biochemical composition and molecular interaction, including water content, hypotonic swelling behavior, and sulfated glycosaminoglycan and collagen contents. In comparison of the top 1-mm slices of talar cartilage with the top 1-mm of tibiofemoral cartilage, the talar cartilage appeared denser with a higher sulfated glycosaminoglycan content, lower water content, higher equilibrium modulus and dynamic stiffness, and lower hydraulic permeability. The equilibrium modulus increased with increasing sulfated glycosaminoglycans per wet weight and decreased with increasing water content for all joint surfaces. Multiple linear regression showed that greater than 80% of the variation in the equilibrium modulus could be accounted for by variations in the biochemical parameters (water content, sulfated glycosaminoglycans/wet weight, and hydroxyproline content/wet weight) for each joint surface. Nonhomogeneous depth-dependent changes in the physical properties and biochemical composition of full-thickness distal femoral cartilage were consistent with previous reports. Since the compressive deformation of cartilage during cyclic loading is confined to the more superficial regions, the differences in properties of the upper regions of the talar compared with tibiofemoral or femoropatellar cartilage may be important in the etiology of osteoarthritis.

Adolescent↗

Polysulphated glycosaminoglycan treatments can mitigate decreases in stiffness of articular cartilage in a traumatized animal joint.

A single, blunt impact to the rabbit patellofemoral joint has been shown to decrease the stiffness of retropatellar cartilage and increase the thickness of the underlying bone. Polysulphated glycosaminoglycan treatments, on the other hand, have been shown to inhibit the degradation of articular cartilage and possibly increase synthesis of collagen and glycosaminoglycans in experimental studies on diseased joints. The aim of the current study was to examine the effect of early treatments with polysulphated glycosaminoglycans on cartilage using an in vivo post-trauma animal model. The study used 24 Flemish Giant rabbits in three groups: control, impacted, and impacted with treatment. Treatment consisted of intramuscular injections the day of insult and every 4 days thereafter for 6 weeks. At 30 weeks after trauma, mechanical tests were performed on the retropatellar cartilage to determine its mechanical stiffness. The patellae were also grossly evaluated for surface lesions on the retropatellar cartilage and histologically processed to measure the thickness of the subchondral bone. The rabbits that received no treatment had a statistically significant decrease in stiffness (modulus) for the cartilage of the impacted patellae compared with that of the contralateral, unimpacted patellae and compared with the cartilage of rabbits in the control group. The degradation in mechanical stiffness, however, was not observed in patellae of rabbits in the group receiving treatment. There was also a significant increase in the underlying thickness of the subchondral plate on the impacted patellae compared with that on the contralateral, unimpacted sides for rabbits in both the treated and nontreated groups. In conclusion, the polysulphated glycosaminoglycan treatments minimized a decrease in mechanical stiffness (modulus) of retropatellar articular cartilage 30 weeks after trauma. The mechanism by which the mechanical stiffness of the cartilage was preserved is unknown.

Animals↗

Composition and transport properties of human ankle and knee cartilage.

The incidence of osteoarthritis is significantly higher in the knee as compared to the ankle, suggesting that differences in the properties of cartilage from these joints may contribute to the development of osteoarthritis. As an avascular tissue, articular cartilage depends primarily upon diffusion for molecular transport. The goal of this study was to determine if differences in the structure and composition between ankle and knee cartilage were also reflected as differences in solute transport properties. The diffusion coefficient and partition coefficient of a 70-kDa dextran molecule were measured in human ankle and knee articular cartilage using fluorescence recovery after photobleaching (FRAP) and were compared to the proteoglycan, collagen, water, and DNA contents within each zone. The mean partition coefficient was significantly lower in the ankle compared to the knee (0.010+/-0.002 vs. 0.022+/-0.003, p<0.01), but no differences in the diffusion coefficients were observed (34.6 +/- 0.9 microm(2)s(-1) vs. 35.4+/-2.4 microm(2)s(-1), p=0.70). Ankle cartilage exhibited higher proteoglycan content as well as a trend toward lower water content, suggesting that ankle cartilage has a smaller effective pore size than knee cartilage. These findings suggest that differences in the composition of ankle and knee cartilage contribute to a difference in the partition coefficient. The results of this study provide further support for the hypothesis that the transport properties of cartilage may play a role in the differences in the incidence of osteoarthritis in these joints by altering the effective concentration of growth factors and cytokines to which chondrocytes are exposed.

Ankle Joint↗

Laser solder welding of articular cartilage: tensile strength and chondrocyte viability.

BACKGROUND AND OBJECTIVE: The surgical treatment of full-thickness cartilage defects in the knee joint remains a therapeutic challenge. Recently, new techniques for articular cartilage transplantation, such as mosaicplasty, have become available for cartilage repair. The long-term success of these techniques, however, depends not only on the chondrocyte viability but also on a lateral integration of the implant. The goal of this study was to evaluate the feasibility of cartilage welding by using albumin solder that was dye-enhanced to allow coagulation with 808-nm laser diode irradiation. STUDY DESIGN/MATERIALS AND METHODS: Conventional histology of light microscopy was compared with a viability staining to precisely determine the extent of thermal damage after laser welding. Indocyanine green (ICG) enhanced albumin solder (25% albumin, 0.5% HA, 0.1% ICG) was used for articular cartilage welding. For coagulation, the solder was irradiated through the cartilage implant by 808-nm laser light and the tensile strength of the weld was measured. RESULTS: Viability staining revealed a thermal damage of typically 500 m in depth at an irradiance of approximately 10 W/cm(2) for 8 seconds, whereas conventional histologies showed only half of the extent found by the viability test. Heat-bath investigations revealed a threshold temperature of minimum 54 degrees C for thermal damage of chondrocytes. Efficient cartilage bonding was obtained by using bovine albumin solder as adhesive. Maximum tensile strength of more than 10 N/cm(2) was achieved. CONCLUSIONS: Viability tests revealed that the thermal damage is much greater (up to twice) than expected after light microscopic characterization. This study shows the feasibility to strongly laser weld cartilage on cartilage by use of a dye-enhanced albumin solder. Possibilities to reduce the range of damage are suggested.

Animals↗

Can MTR be used to assess cartilage in the presence of Gd-DTPA2-?

Magnetization transfer (MT) and T(1) and T(2) relaxation of normal, trypsinized, and interleukin-1beta (IL-1beta)-treated cartilage were measured in the absence and presence of Gd-DTPA(2-). Without the addition of Gd-DTPA(2-), neither T(1) nor T(2) showed any significant change with cartilage damage. However, with Gd-DTPA(2-), trypsinized cartilage exhibited substantially shorter T(1) than normal cartilage, as expected due to the glycosaminoglycan (GAG) loss in these samples, and associated increased Gd-DTPA(2-) concentration. The T(2) results were similar, but less dramatic. The MT pseudo first-order exchange rate, RM(0B), did not depend on the contrast agent concentration, as expected, and was significantly faster for trypsinized and slower for IL-1beta-treated cartilage. In both cases, the MT fraction of the macromolecular pool M(0B) decreased while only trypsinized cartilage showed an increase in MT exchange rate R. The MT ratio (MTR) decreased with increasing Gd-DTPA(2-) concentration. However, interpretation of the MTR results in the presence of Gd-DTPA(2-) was complicated due to competing effects of increased longitudinal relaxivity and MT exchange. Therefore, in a cartilage sample with an unknown degree of GAG depletion and some collagen damage, a full MT analysis might be used to probe the molecular state of cartilage, but it would not be possible to use a simple MTR measurement after the administration of Gd-DTPA(2-) to differentially determine the amount of cartilage degradation in the sample.

Animals↗

Short TE MR microscopy: accurate measurement and zonal differentiation of normal hyaline cartilage.

The purpose of this study was to use MR imaging to accurately measure the thickness of hyaline cartilage and determine the MR contrast parameters for differentiation of cartilage zones in normal human cartilage samples. Cartilage samples were examined using three dimensional spin-echo MR microscopy at 9.4 T with a voxel size of 31 x 31 x 300 microns. Effects of T2 signal loss, susceptibility, and partial volume on measured thickness of cartilage were investigated. Thickness measurements were obtained on corresponding histological sections for comparison. Optimal contrast parameters for delineation of cartilage zones were evaluated using magnetization transfer, inversion recover, T1, and T2 contrast. T2 relaxation losses were identified as the primary source of discrepancy between the measured thickness of cortical bone and hyaline cartilage. Good contrast for zonal differentiation was obtained using T1 weighting. We conclude that images obtained using short TE MR microscopy can be used to accurately measure cartilage and bone thickness in human specimens, and can demonstrate zones within normal cartilage.

Bone and Bones↗

In vivo precision of quantitative shoulder cartilage measurements, and changes after spinal cord injury.

Recent advances in MRI have enabled the quantitative assessment of articular cartilage morphology in human joints. In this study, we tested the hypothesis that the precision of quantitative shoulder cartilage measurements is sufficient to detect changes between and within patients, and that shoulder cartilage thickness in paraplegic patients increases due to increased loading. We imaged the shoulders of seven healthy volunteers four times using a coronal 3D, fat-suppressed, gradient-echo sequence. The humeral head cartilage in seven paraplegic patients was evaluated soon after injury and 1 year post injury. A precision of 4.5% (root mean square (RMS) average coefficient of variation (CV) %) was found for shoulder cartilage thickness measurements in the humeral head. Whereas a significant decrease of cartilage thickness (-11%, P < 0.05) was observed in the knee, there was no significant change in articular cartilage thickness in the shoulder (-1.1%). Our data show, for the first time, that articular cartilage of the humeral head can be quantified with acceptable precision in vivo. It was demonstrated that, in contrast to the knee, the articular cartilage morphology of the humeral head changes very little (i.e., there is no significant increase or decrease in thickness) after spinal cord injury (SCI).

Adaptation, Physiological↗

Collagen dynamics in articular cartilage under osmotic pressure.

Cartilage is a complex biological tissue consisting of collagen, proteoglycans and water. The structure and molecular mobility of the collagen component of cartilage were studied by (13)C solid-state NMR spectroscopy as a function of hydration. The hydration level of cartilage was adjusted between fully hydrated ( approximately 80 wt% H(2)O) and highly dehydrated ( approximately 30 wt% H(2)O) using the osmotic stress technique. Thus, the conditions of mechanical load could be simulated and the response of the tissue macromolecules to mechanical stress is reported. From the NMR measurements, the following results were obtained. (i) Measurements of motionally averaged dipolar (1)H-(13)C couplings were carried out to study the segmental mobility in cartilage collagen at full hydration. Backbone segments undergo fast motions with amplitudes of approximately 35 degrees whereas the collagen side-chains are somewhat more mobile with amplitudes between 40 and 50 degrees . In spite of the high water content of cartilage, collagen remains essentially rigid. (ii) No chemical shift changes were observed in (13)C cross-polarization magic angle spinning spectra of cartilage tissue at varying hydration indicating that the collagen structure was not altered by application of high osmotic stress. (iii) The (1)H-(13)C dipolar coupling values detected for collagen signals respond to dehydration. The dipolar coupling values gradually increase upon cartilage dehydration, reaching rigid limit values at approximately 30 wt% H(2)O. This indicates that collagen is essentially dehydrated in cartilage tissue under very high mechanical load, which provides insights into the elastic properties of cartilage collagen, although the mechanical pressures applied here exceed the physiological limit.

Animals↗

Effects of retinoic acid on cartilage differentiation in a chondrogenic cell line.

Previously we have isolated the monopotential chondrogenic cell line RCJ 3.1 C5.18 from the multipotential mesenchymal cell line RCJ 3.1 [Grigoriadis et al.: Endocrinology, 125:2103-2110, 1989]. When cultured for approximately 20 days under appropriate conditions, these cells from cartilage nodules. In the present investigation, we have used this cell line to study the effects of all-trans retinoic acid (RA) on chondroblast differentiation, cartilage formation, and cartilage degradation. Continuous exposure of cultures to RA (0.01-100 nM) inhibited chondroblast differentiation and glycosaminoglycan (GAG) accumulation in a dose-dependent manner, without comparable effects on cell growth. Pulse treatment with RA for various 4 day periods during a 17-24 day culture period established that RA inhibited differentiation of chondroprogenitors at all periods tested. These effects were reversible, except for part of the effect on early chondroprogenitors. Treatment with RA on days 13-17 in 17 day cultures not only resulted in cessation of cartilage formation, but also in disappearance of pre-existing cartilage nodules. We demonstrated that this was associated with RA-induced downregulation of GAG synthesis and increased degradation of cartilage proteoglycans. Hence, the inhibitory effects of RA on cartilage formation consist of inhibition of chondroblast differentiation, inhibition of GAG synthesis by differentiated chondroblasts, and stimulation of cartilage proteoglycan degradation by differentiated chondroblasts and/or chondrocytes. These results indicate that the clonal monopotential chondrogenic cell line RCJ 3.1 C5.18 forms a good model system to study the effects of retinoids on cartilage differentiation, formation, and degradation.

Animals↗

Differential effects of bFGF and IGF-I on matrix metabolism in calf and adult bovine cartilage explants.

The effects of basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) on cell and matrix metabolism in calf and adult bovine cartilage explants were examined. In calf cartilage, bFGF elicited dose-dependent and bi-directional effects on mitotic activity and anabolic processes. Addition of bFGF at 3 ng/ml stimulated cell mitotic activity (total DNA) and synthesis of proteoglycan ([35S]sulfate incorporation), protein ([3H]proline incorporation), and collagen (formation of [3H]hydroxyproline), and resulted in a slight increase in proteoglycan deposition compared to basal medium. However, 30-300 ng/ml of bFGF inhibited mitotic activity and synthetic processes, accelerated [35S]proteoglycan release compared to basal medium, and resulted in an inhibition of proteoglycan deposition during the culture period. In contrast, treatment of adult cartilage with 3-300 ng/ml of bFGF did not affect the DNA content but did stimulate synthetic processes in a dose-dependent manner. Basic FGF also had bidirectional effects on matrix catabolism in adult cartilage, with 3 ng/ml accelerating [35S]proteoglycan release, but 30-300 ng/ml of bFGF resulting in release rates comparable to that in basal medium. Nonetheless, even with maximal bFGF stimulation, adult bovine cartilage suffered a net loss of proteoglycan during culture. Addition of 3-300 ng/ml of IGF-I to either calf or adult bovine cartilage stimulated synthetic processes and shifted the metabolic balance toward a net deposition of proteoglycan. Neither bFGF nor IGF-I altered the low basal rate of [3H]hydroxyproline release from either calf or adult bovine cartilage. Thus, (i) the regulatory effects of bFGF and IGF-I on bovine articular cartilage appear age-dependent, and (ii) bFGF is capable of promoting either anabolic or catabolic processes, and may therefore serve a dual role in the regulation of cartilage metabolism.

Aging↗

Association of proteoglycan degradation with catabolic cytokine and stromelysin release from cartilage cultured with fibronectin fragments.

Addition of fibronectin fragments to bovine articular cartilage explant cultures results in enhanced release of metalloproteinases and rapid cartilage proteoglycan (PG) degradation and loss. The chondrolysis begins with rapid PG degradation which markedly slows after 1 week. Preliminary observations suggest that catabolic cytokines mediate chondrolytic activities of the fibronectin fragments. The objectives of this work were to investigate the correlations between: (a) release of specific cytokines; (b) release of the metalloproteinase (MMP), stromelysin-1 (MMP-3); (c) release of the tissue inhibitor of MMPs, TIMP-1, and; (d) degradation and release of PG from cultured cartilage. We report that human articular cartilage cultured with an amino-terminal 29-kDa fragment (Fn-f) at 0.1 microM, released enhanced levels of TNF-alpha, IL-1beta, and IL-1alpha with peaks at Days 2, 3, and 9, respectively. MMP-3 release was elevated with a peak at Day 6 and a profile similar to that for the Fn-f-induced cartilage PG depletion. IL-6 release was enhanced within 2 days and continued at the same level throughout the culture period but this did not lead to enhanced release of TIMP-1, a known activity of IL-6. These data suggest that in the early chondrolytic events induced in cultured cartilage by Fn-f, enhanced MMP-3 release and maximal degradation and release of PG from cultured cartilage are kinetically associated with elevated release of the catabolic cytokines, TNF-alpha, IL-1beta, and IL-1alpha. Further, a later period of slowing PG loss and slowing MMP-3 release is associated with greatly slowed release of these cytokines, but prolonged release of IL-6. This model of cartilage damage may be useful for studies of the interplay between cytokines and the effects of combinations of cytokines on cartilage homeostasis.

Aged↗