Cartilage Homogenates Research Articles

Comparative analysis of the degree of hydrolysis of biopolymers in hyaline cartilage homogenates in the presence of proteolytic enzymes

Investigating the degree of hydrolysis in the presence of different proteolytic enzymes is one of the tasks regarding further development of nutraceuticals obtained from connective tissues for biomedicine. Hydrolysis of biopolymers in cattle and swine tracheal hyaline cartilage homogenates was carried out in the presence of the following enzymes: pancreatin, chymopsin, papain and karipazim, a drug that contains papain, a proteolytic enzyme. This study shows that karipazim manufactured by MedFlorina LLC has better efficacy than karipazim manufactured by ZAO Vifiteh, and the greatest degree of hydrolysis for collagen occurrs at 60°C and karipazim concentration of 10%. More complete hydrolysis of proteoglycans was performed in the cattle hyaline cartilage homogenates, because the analysis of NMR spectra revealed the glucose, the final product of glycosaminoglycan hydrolysis.

Comparative Analysis of the Degree of Hydrolysis of Biopolymers in Hyaline Cartilage Homogenates in the Presence of Proteolytic Enzymes

Comparative Analysis of the Degree of Hydrolysis of Biopolymers in Hyaline Cartilage Homogenates in the Presence of Proteolytic Enzymes

Isoprenaline and salbutamol inhibit pyroptosis and promote mitochondrial biogenesis in arthritic chondrocytes by downregulating β-arrestin and GRK2.

Rheumatoid arthritis and osteoarthritis overlap many molecular mechanisms of cartilage destruction. Wear and tear in cartilage is chondrocyte-mediated, where chondrocytes act both as effector and target cells. In current study, role of β2-AR was studied in chondrocytes both in vitro and in vivo. High grade inflammation in vitro and in vivo disease models led to decline in anti-inflammatory β2-AR signaling and use of β2-AR agonist attenuated arthritis symptoms. Detailed analysis in chondrocytes revealed that Isoprenaline (ISO) and Salbutamol (SBT) increased cell viability and relative Bcl-2 expression, meanwhile, decreased proteins levels of TNF-α, IL-6 and IL-8 in arthritic chondrocytes when compared with control, respectively. SBT preserved physiological concentration of antioxidant enzymes (CAT, POD, SOD and GSH) in cartilage homogenates and ISO inhibited IL-1β-mediated genotoxicity in arthritic chondrocytes. Moreover, β2-AR agonist increased mitochondrial biogenesis and proteoglycan biosynthesis by upregulating the gene expression of PGC1-α, NRF2 and COL2A1, Acan, respectively. ISO and SBT inhibited extracellular matrix (ECM) degradation by downregulating the gene expression of MMP1, MMP3, MMP9 and ADAMTS5 in vitro and in vivo study. In mechanism, β2-AR agonists decreased β-arrestin and GRK2 pathway, and as a result mice receiving SBT did not exhibit severe disease. Hence our data suggest β2-AR agonist administered at disease onset can inhibit receptor internalization by downregulating the expression of β-arrestin and GRK2 in chondrocytes.

Efficacy of Combination Therapy with Apigenin and Synovial Membrane-Derived Mesenchymal Stem Cells on Knee Joint Osteoarthritis in a Rat Model.

Background: Osteoarthritis (OA) is a degenerative joint disease that causes a variety of adverse health effects. Considering the need to identify additional effective therapeutic options for OA therapy, we investigated the effect of co-injection of apigenin and synovial membrane-derived mesenchymal stem cells (SMMSCs) on OA in male rats’ knee joints.Methods: The study was performed in 2019 at the Department of Pharmacology, Shiraz University of Medical Sciences, Shiraz, Iran. Anterior cruciate ligament transection (ACLT) was used to induce OA. For three weeks, male Sprague-Dawley rats (eight groups, n=6 each) were treated once-weekly with intra-articular injections of apigenin alone or in combination with SMMSC (three million cells), phosphate-buffered saline, or hyaluronic acid. After three months, the interleukin 1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), superoxide dismutase (SOD), and malondialdehyde (MDA) levels were measured in the cartilage homogenate. The expression of extracellular matrix (ECM) components including collagen 2a1, aggrecan, IL-1β, TNF-α, inducible nitric oxide synthase (iNOS), transcription factor SOX-9, and matrix metalloproteinases 3 and 13 were assessed using real-time polymerase chain reaction (RT-PCR) analysis. Radiological evaluation and histopathological assessment were used to evaluate the knees.Results: Levels of TNF-α (P=0.009), MDA (P>0.001), and IL-1β (P<0.001) decreased and the level of SOD increased (P=0.004) in the apigenin 0.3 µM with SMMSCs group. RT-PCR analysis indicated that IL-1β in the apigenin 0.3 µM with SMMSCs group reduced significantly (P<0.001). This group also exhibited increased expression levels of SOX-9, collagen 2a1, and aggrecan (P<0.001).Conclusion: Apigenin may have supplementary beneficial effects on cell therapy in a rat model of OA due to its possible effect on the reduction of oxidative stress, suppression of inflammation, and promotion of production of ECM components.

Preparation of Type II Collagen Short Peptides: Temperature Conditions of Cartilage Homogenization and Collagen Hydrolysis.

Physicochemical properties of hyaline cartilage homogenates were studied by the method of microcalorimetry. Collagen hydrolysates were obtained after homogenization of hyaline cartilages under high pressure conditions at the temperatures that denaturate collagen. Thermodynamic parameters of thermal transition of collagen in cartilage suspension were determined. Enthalpy of thermal transition ΔН decreases in comparison with the control. Thermal transition half-width ΔТ varies with temperature. More denatured and homogeneous samples were obtained at homogenization temperature 80°C. According to spectral studies, particles in the samples obtained at the temperature of 80°C were smaller. The temperature of 80°C is preferred for homogenizing hyaline cartilages and obtaining collagen type II short peptides.

Получение коротких пептидов коллагена II типа: температурные условия гомогенизации хрящей и гидролиз коллагена

Physicochemical properties of hyaline cartilage homogenates were studied by the method of microcalorimetry. Collagen hydrolysates were obtained after homogenization of hyaline cartilages under high pressure conditions at the temperatures that denaturate collagen. Thermodynamic parameters of thermal transition of collagen in cartilage suspension were determined. Enthalpy of thermal transition ΔН decreases in comparison with the control. Thermal transition half-width ΔТ varies with temperature. More denatured and homogeneous samples were obtained at homogenization temperature 80°C. According to spectral studies, particles in the samples obtained at the temperature of 80°C were smaller. The temperature of 80°C is preferred for homogenizing hyaline cartilages and obtaining collagen type II short peptides.

The effect of kaempferol and apigenin on allogenic synovial membrane-derived stem cells therapy in knee osteoarthritic male rats

BackgroundBased on the anti-inflammatory and anti-oxidant properties of kaempferol and apigenin, we hypothesized that co-injection of these phytochemicals would increase the effectiveness of cell therapy in knee osteoarthritic rats. MethodsAnterior cruciate ligament transection (ACLT) was used to induce osteoarthritis (OA). Animals were treated by weekly intra-articular injections of kaempferol (10 or 20 μM) and/or isolated MSCs from synovial membrane (SMMSCs) (3 × 106 cells), a mixture of apigenin (0.1 μM) and kaempferol alone or SMMSCs, hyaluronic acid or PBS (group size n = 6), for three weeks. After three months, the levels of IL-1β, tumor necrosis factor alpha (TNF-α), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured in the cartilage homogenate. Furthermore, relative expressions of collagen II2a1, aggrecan, IL-1β, TNF-α, inducible nitric oxide synthase (iNOS), SOX-9, MMP-3 and MMP-13 were assessed using real-time PCR. Radiological evaluation, before/after treatments, and histopathological assessments were carried out to evaluate the knees. ResultsNon-toxic concentrations of kaempferol and apigenin determined to be 10, 20 μM and 0.1, 0.3 μM, respectively. In comparison with the OA group, the levels of TNF-α, IL-1β and MDA significantly decreased in OA + MSCs + kaempferol + apigenin group and a significant increase in SOD level was observed. The levels of MMP-13, MMP-3, TNF-α, IL-1β, iNOS were significantly decreased in the groups of OA + MSCs + A0.1 μM + K10 μM and OA + MSCs + K20 μM. Co-treatment of kaempferol and apigenin increased the gene expression levels of collagen IIa1, aggrecan and SOX-9 genes. ConclusionWe showed that kaempferol and apigenin potentially increase the efficiency of OA cell therapy in the rat model of ACLT-induced OA.

Crucial role of the terminal complement complex in chondrocyte death and hypertrophy after cartilage trauma

Innate immune response and particularly terminal complement complex (TCC) deposition are thought to be involved in the pathogenesis of posttraumatic osteoarthritis. However, the possible role of TCC in regulated cell death as well as chondrocyte hypertrophy and senescence has not been unraveled so far and was first addressed using an ex vivo human cartilage trauma-model. Cartilage explants were subjected to blunt impact (0.59J) and exposed to human serum (HS) and cartilage homogenate (HG) with or without different potential therapeutics: RIPK1-inhibitor Necrostatin-1 (Nec), caspase-inhibitor zVAD, antioxidant N-acetyl cysteine (NAC) and TCC-inhibitors aurintricarboxylic acid (ATA) and clusterin (CLU). Cell death and hypertrophy/senescence-associated markers were evaluated on mRNA and protein level. Addition of HS resulted in significantly enhanced TCC deposition on chondrocytes and decrease of cell viability after trauma. This effect was potentiated by HG and was associated with expression of RIPK3, MLKL and CASP8. Cytotoxicity of HS could be prevented by heat-inactivation or specific inhibitors, whereby combination of Nec and zVAD as well as ATA exhibited highest cell protection. Moreover, HS+HG exposition enhanced the gene expression of CXCL1, IL-8, RUNX2 and VEGFA as well as secretion of IL-6 after cartilage trauma. Our findings imply crucial involvement of the complement system and primarily TCC in regulated cell death and phenotypic changes of chondrocytes after cartilage trauma. Inhibition of TCC formation or downstream signaling largely modified serum-induced pathophysiologic effects and might therefore represent a therapeutic target to maintain the survival and chondrogenic character of cartilage cells.

Human Cartilage Homogenates Influence the Crystallization of Monosodium Urate and Inflammatory Response to Monosodium Urate Crystals: A Potential Link Between Osteoarthritis and Gout.

Monosodium urate (MSU) crystal deposition and gout flares frequently affect osteoarthritic joints. This study was undertaken to examine the effects of human cartilage homogenates on MSU crystallization and MSU crystal-induced inflammation. Human cartilage homogenates were prepared from macroscopically healthy and macroscopically diseased knee joint samples. Crystallization assays were used to test the effects of cartilage homogenates or individual cartilage factors on MSU crystallization. Changes in urate solubility, crystal nucleation, crystal growth, and total crystal mass were determined. THP-1 cell assays were used to assess cytokine release following culture with MSU crystals grown in the presence or absence of cartilage homogenates or individual proteins. Addition of either 5% or 10% healthy cartilage homogenate increased the total mass of MSU crystals formed and resulted in formation of shorter MSU crystals compared to controls without cartilage homogenate. MSU crystal bows were observed in both the presence and absence of cartilage homogenate; however, bows formed in the presence of cartilage homogenates were significantly shorter than bows formed in their absence. There were no effect differences between macroscopically healthy and macroscopically diseased cartilage homogenates in all assessments. Addition of either type II collagen or albumin also led to the formation of shorter MSU crystals. In THP-1 cell assays, MSU crystals grown with healthy cartilage homogenate increased the release of interleukin-8, whereas MSU crystals grown with type II collagen or albumin had no effect on inflammatory cytokine release. In the presence of elevated urate levels, human cartilage homogenates increase MSU crystal formation and promote the formation of smaller crystals, which have greater inflammatory potential. These processes may contribute to the predilection of osteoarthritic joints to develop gout.

Factors influencing the crystallization of monosodium urate: a systematic literature review.

BackgroundGout is a chronic disease of monosodium urate (MSU) crystal deposition. Although hyperuricaemia is the central risk factor for development of gout, not all people with hyperuricaemia have subclinical MSU crystal deposition or indeed, symptomatic disease. The aim of this systematic literature review was to identify factors that contribute to MSU crystallization.MethodsA search was conducted of the electronic databases PubMed, Science Direct and Scopus. Articles were included if they contained original data related to MSU crystallization. The methods and results were summarized and categorized into articles describing at least one of the three key steps in MSU crystallization (reduced urate solubility, nucleation and growth).ResultsA total of 2175 articles were initially identified in our systematic search with 35 of these articles included in the final analysis. Elevated urate concentration was identified as a central factor driving all three stages of MSU crystallization. Factors that were found to consistently reduce urate solubility were reduced temperatures, pH 7–9 and various ions including sodium ions. Connective tissue factors including bovine cartilage homogenates and healthy human synovial fluid and serum all enhanced urate solubility. MSU nucleation was found to be increased by a number of factors, including sodium ions, uric acid binding antibodies, and synovial fluid or serum from patients with gout. Other than elevated urate concentrations, no other specific factors were identified as promoters of MSU crystal growth.ConclusionsIncreased urate concentration is the key factor required at each stage of MSU crystallization. Different proteins and factors within connective tissues may promote MSU crystallization and may be important for determining the sites at which MSU crystallization occurs in the presence of elevated urate concentrations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12891-015-0762-4) contains supplementary material, which is available to authorized users.

Human monoclonal antibody fragments targeting matrilin-3 in growth plate cartilage.

Many genetic disorders, including chondrodysplasias, and acquired disorders impair growth plate function, resulting in short and sometimes malformed bones. There are multiple endocrine and paracrine factors that promote chondrogenesis at the growth plate, which could potentially be used to treat these disorders. Targeting these growth factors specifically to the growth plate might augment the therapeutic skeletal effect while diminishing undesirable effects on non-target tissues. Using yeast display technology, we selected single-chain variable antibody fragments that bound to human and mouse matrilin-3, an extracellular matrix protein specifically expressed in cartilage tissue. The ability of the selected antibody fragments to bind matrilin-3 and to bind cartilage tissue in vitro and in vivo was assessed by ELISA and immunohistochemistry. We identified antibody fragments that bound matrilin-3 with high affinity and also bound with high tissue specificity to cartilage homogenates and to cartilage structures in mouse embryo sections. When injected intravenously in mice, the antibody fragments specifically homed to cartilage. Yeast display successfully selected antibody fragments that are able to target cartilage tissue in vivo. Coupling these antibodies to chondrogenic endocrine and paracrine signaling molecules has the potential to open up new pharmacological approaches to treat childhood skeletal growth disorders.

Gelatinase activities and haptoglobin concentrations in healthy and in degenerative articular cartilage of pigs

The objectives of this study were to investigate the activity of the matrix metalloproteinase (MMP)-2 and MMP-9 in healthy and in degenerative cartilage and to characterize the relation with the acute phase protein haptoglobin (HP) in articular cartilages of pigs. Joint surfaces of the proximal and distal humerus and femur of fattening pigs were histopathologically classified. In addition, cartilage homogenates and synovia were obtained. The tissue homogenates were analysed for gelatinase activity by zymography and by activity assay. The concentrations of HP in cartilage homogenates, in synovia and in serum were analysed by ELISA. High enzymatic activity of the MMP-2 latent form was observed in zymography in all samples. Zymographic activities of MMP-2 active form and MMP-9 (active and latent form) were detected at low levels in some samples. Comparison of the zymographic activities of gelatinases in unaltered vs. altered cartilages yielded no differences. In contrast to zymography, cartilage homogenates were negative for MMP-2 and MMP-9 in the activity assays. The concentrations of HP in cartilage homogenates and in synovia from samples without alteration and from samples with massive alterations were not different. When classified according to their HP concentration, cartilage homogenates with increased HP concentrations had higher (p < 0.05) zymographic activities of the MMP-2 active form. For the two MMPs investigated, there was no detectable relationship with degenerative processes in the cartilage.

Osteoarthritic Cartilage Is More Homogeneous Than Healthy Cartilage: Identification of a Superior Region of Interest Colocalized With a Major Risk Factor for Osteoarthritis

Cartilage loss as determined by magnetic resonance imaging (MRI) or joint space narrowing as determined by x-ray is the result of cartilage erosion. However, metabolic processes within the cartilage that later result in cartilage loss may be a more sensitive assessment method for early changes. Recently, it was shown that cartilage homogeneity visualized by MRI representing the biochemical changes undergoing in the cartilage is a potential marker for early detection of knee osteoarthritis (OA) and is also able to significantly separate groups of healthy subjects from those with OA. The purpose of this study was twofold. First, we wished to evaluate whether the results on cartilage homogeneity from the previous study can be reproduced using an independent population. Second, based on the homogeneity framework, we present an automatic technique that partitions the region of interest in the cartilage that contributes most to discrimination between healthy and OA subjects and allows for identification of the most implicated areas in early OA. These findings may allow further investigation of whether cartilage homogeneity reveals a predisposition for OA or whether it evolves as a consequence to disease and thereby can be used as a progression biomarker.A total of 283 right and left knees from 159 subjects aged 21 to 81 years were scanned using a Turbo 3D T1 sequence on a 0.18-T MRI Esaote scanner. The medial compartment of the tibial cartilage sheet was segmented using a fully automatic voxel classification scheme based on supervised learning. From the segmented cartilage sheet, homogeneity was quantified by measuring entropy from the distribution of signal intensities inside the compartment. Each knee was examined by radiography, and the knees were categorized by the Kellgren and Lawrence (KL) Index. Next, based on a gradient descent optimization technique, the cartilage region that contributed to the maximum statistical significance of homogeneity in separating healthy subjects from the diseased was partitioned. The generalizability of the region was evaluated by testing for overfitting. Three different regularization techniques were evaluated for reducing overfitting errors.The P values for separating the different groups based on cartilage homogeneity were 2 x 10(-5) (KL 0 versus KL 1) and 1 x 10(-7) (KL 0 versus KL >0). Using the automatic gradient descent technique, the partitioned region was toward the peripheral part of the cartilage sheet. Using this region, the P values for separating the different groups based on homogeneity were 5 x 10(-9) (KL 0 versus KL 1) and 1 x 10(-15) (KL 0 versus KL >0). The precision of homogeneity for the partitioned region assessed as a test-retest root-mean-square coefficient of variation was 3.3%. Bootstrapping proved to be an effective regularization tool in reducing overfitting errors.The validation study supported the use of cartilage homogeneity as a tool for the early detection of knee OA and for separating groups of healthy subjects from those who have disease. Our automatic, unbiased partitioning algorithm based on a general statistical framework outlined the cartilage region of interest that best separated healthy from OA conditions on the basis of homogeneity discrimination. We have shown that OA affects certain areas of the cartilage more distinctly, and these areas are located more toward the peripheral region of the cartilage. We propose that this region corresponds anatomically to cartilage covered by the meniscus in healthy subjects. This finding may provide valuable clues in the early detection and monitoring of OA and thus may improve treatment efficacy.

Separation of healthy and early osteoarthritis by automatic quantification of cartilage homogeneity

Cartilage loss as determined either by magnetic resonance imaging (MRI) or by joint space narrowing in X-rays is the result of cartilage erosion. However, metabolic processes within the cartilage that later result in cartilage loss may be a more accurate assessment method for early changes. Early biological processes of cartilage destruction are among other things, a combination of proteoglycan turnover, as a result of altered charge distributions, and local alterations in water content (edema). As water distribution is detectable by MRI, the aim of this study was to investigate cartilage homogeneity visualized by MRI related to water distribution, as a potential very early marker for early detection of knee osteoarthritis (OA). One hundred and fourteen right and left knees from 71 subjects aged 22-79 years were scanned using a Turbo 3D T(1) sequence on a 0.18T MRI Esaote scanner. The medial compartment of the tibial cartilage sheet was segmented using a fully automatic voxel classification scheme based on supervised learning. From the segmented cartilage sheet, homogeneity was quantified by measuring entropy from the distribution of signal intensities inside the compartment. For each knee an X-ray was acquired and the knees were categorized by the Kellgren and Lawrence (KL) index and the joint space width (JSW) was measured. The P-values for separating the groups by each of JSW, cartilage volume, cartilage mean intensity, and cartilage homogeneity were calculated using the unpaired t-test. The P-value for separating the group diagnosed as KL 0 from the group being KL 1 based on JSW, volume and mean signal intensity the values were P=0.9, P=0.4 and P=0.0009, respectively. In contrast, the P-value for homogeneity was P=0.0004. The precision of the measures assessed, as a test-retest root mean square coefficient of variation (RMS-CV%) was 3.9% for JSW, 7.4% for volume, 3.9% for mean signal intensity and 3.0% for homogeneity quantification. These data demonstrate that the distribution of components of the articular matrix precedes erosion, as measured by cartilage homogeneity related to water concentration. We show that homogeneity was able to separate early OA from healthy individuals in contrast to traditional volume and JSW quantifications. These data suggest that cartilage homogeneity quantification may be able to quantify early biochemical changes in articular cartilage prior to cartilage loss and thereby provide better identification of patients for OA trials who may respond better to medicinal intervention of some treatments. In addition, this study supports the feasibility of using low-field MRI in clinical studies.

Distribution of chondroitin 4-sulfate epitopes (2/B/6) in various zones and compartments of articular cartilage in guinea pig osteoarthrosis.

We studied changes in the morphology of tibial articular cartilage in early guinea pig osteoarthrosis (OA) at 6 and 12 months of age with quantitative light microscopy, and the distribution of chondroitin-4-sulfate with quantitative ultrastructural immunolabeling, using the 2/B/6 epitope. Labeling was correlated to previous chromatography findings concerning proteoglycan (PG) concentration in animals of the same age. The cell volume fraction had decreased at 12 months in the superficial zone of cartilage with OA (medial condyle) as well as in cartilage without OA (lateral condyle), being lower medially than laterally. The PG concentration differed between the zones and matrix compartments. Medially, a reduction in PG concentration occurred between 6 and 12 months in the interterritorial compartment of the two uppermost zones. Laterally, the concentrations increased. In general, the pericellular PG concentration was higher than the interterritorial in the two uppermost zones. The striking variation in structural and labeling responses in the various zones and compartments indicate a heterogeneous tissue response in guinea pig OA that will probably affect sampling in biochemical analyses of cartilage homogenates and synovial fluid.

Staphylococcus aureus stimulates inducible nitric oxide synthase in articular cartilage.

To determine if Staphylococcus aureus stimulates the L-arginine-nitric oxide (NO) synthase pathway in articular cartilage. A heat-killed and sonicated (denatured) S. aureus preparation was added to cultures of bovine articular cartilage. NO production was measured as accumulated nitrite in the culture medium and by the NO synthase-dependent conversion of 3H-L-arginine to 3H-L-citrulline in cartilage homogenates. Inducible NO synthase (iNOS) messenger RNA (mRNA) expression was analyzed by Northern blot. Proteoglycan synthesis was measured by 35SO4 incorporation into glycosaminoglycan. Nitrite accumulation and 3H-L-citrulline formation in cartilage were elevated by denatured S. aureus (compared with unstimulated control cartilage) and inhibited by the NO synthase inhibitor N(G)-monomethyl-L-arginine. Northern blot analysis revealed increased iNOS mRNA expression in bovine chondrocytes in response to denatured S. aureus stimulation. Denatured S. aureus suppressed the accumulation of 35SO4-labeled macromolecules representing newly synthesized proteoglycans in bovine articular cartilage. The suppressed proteoglycan synthesis was due to the presence of NO. These findings support the hypothesis that a component of S. aureus can stimulate iNOS in articular cartilage, and that NO generated from this enzyme down-regulates cartilage matrix proteoglycan synthesis.

Specificity of a porcine 127‐kd nucleotide pyrophosphohydrolase for articular tissues

To determine the tissue specificity of a porcine 127-kd nucleotide pyrophosphohydrolase (NTPPHase) found in vesicles derived from hyaline articular cartilage (ACV). Homogenates of porcine brain, lung, liver, kidney, urinary bladder, pancreas, spleen, skin, vena cava, marrow, bone (cells), tendon (Achilles), ligament (anterior cruciate), elastic cartilage, meniscus, and hyaline cartilage were analyzed for NTTPHase activity (thymidine monophosphate paranitrophenyl ester substrate) and by Western blot using polyclonal antibodies against 127-kd NTPPHase and against recombinant PC-1, another ecto-NTPPHase. All tissues contained NTPPHase activity; the highest specific activity was found in hyaline articular cartilage, the lowest in brain. ACV-associated 127-kd NTTPHase was expressed in cartilage, ligament, and tendon. PC-1 was also expressed in those tissues and in skin, kidney, bone cells, and (probably) in liver and muscle. The 127-kd NTPPHase appears to be highly specific for articular tissues.

Specific β Estradiol Binding in Cartilage and Serum from Young Mice and Rats is Age Dependent

Various studies have shown a direct effect of beta estradiol on cartilage and bone. Such effects point to the possibility that specific receptors to estradiol exist in the growth plate cartilage as well as in bone. 3H-estradiol specific binding (EB) was therefore investigated in the supernatant of cartilage homogenates from the epiphyses and ribs of young growing mice and rats. High levels of EB were observed in the cytosol fraction of cartilage homogenates in the late fetal stage and in young rats and mice. The EB levels decreased gradually from late fetal stage up to 14 days of age in both groups of animals independent of their sex. Nuclear binding of 3H estradiol was also demonstrated by autoradiography in the chondrocytes of proliferating and hypertrophic zones. Estradiol binding was inhibited by high doses of unlabelled beta-estradiol, but not by alpha estradiol. Binding was also inhibited by tamoxifen and DES but not by testosterone. High levels of estradiol binding (EBS) were also observed in serum from young animals, but not in animals 2 months of age or older. Study of estradiol binding in cartilage and in serum of rats of the same age showed a significant difference in estradiol binding between these two systems. The difference in estradiol binding between serum and cartilage was seen in the response to inhibitors, Scatchard analysis, and temperature dependence. The results of our study imply that there are specific receptors for 17 beta estradiol in growth plate cartilage; they originate from chondrocytes, and their amount decreases with age. The effects of estradiol on endochondral bone growth seems therefore to be receptor mediated.

Isolated chondrons: a concept comes of age!

The chondron concept was first introduced in 1925 by Benninghoff who used polarized light microscopy to show that the chondrocyte in hyaline cartilages was surrounded by a specialized region of matrix. However, this concept was not widely accepted at the time, and it was until 1969 that Szirmai rekindled interest in the chondron concept. Using new histochemical interpretations coupled with microchemical analysis, Szirmai showed the micro-world of the chondron consisted of a fine collagenous perilacunar rim surrounding a proteoglycan rich lacunar space. He also showed that chondrons could be extracted from horse nasal septum by high speed homogenization and concluded that the chondron was mechanically robust and represented the primary functional and metabolic unit of hyaline cartilages. Again the chondron concept was not widely embraced despite the rapid accumulation of data indicating common specializations in the vicinity of hyaline cartilage chondrocytes. In 1985, our own research on the structure and function of adult articular cartilage matrices led to the discovery of chondrons in the homogenate produced by slow speed homogenization of canine tibial cartilage. The isolation of intact chondrons conclusively established the chondron as a microanatomical unit of adult articular cartilage. Each chondron consisted of a chondrocyte intimately associated with a proteoglycan rich pericellular matrix which was surrounded by a densely compacted pericellular capsule composed of fine collagen species. We have now developed the isolated chondron as a model to study the natural interrelationship between the chondrocyte and its pericellular microenvironment. Using morphological, histochemical, ultrastructural, immunohistochemical, biochemical and metabolic techniques, we have successfully shown the isolated chondron is rich in chondroitin sulfate, keratan sulfate and hyaluronan glycosaminoglycans, collagen types II, VI, IX, and XI, and glycoproteins such as fibronectin and possibly laminin. Further studies of chondrons extracted from osteoarthritic cartilage suggest the chondron is remodeled during the degenerative process causing the pericellular microenvironment to expand and the chondrocyte to proliferate into the large clusters typical of osteoarthritic pathology. The data presented in this short review indicates that the chondron is a highly specialized microanatomical unit of adult articular cartilage and plays a fundamental role in the initiation and progression of degenerative arthritis. Future studies on the isolated chondron model will ensure a highly greater understanding of the role of the cellular microenvironment in articular cartilage biology and pathology. Biomedical Reviews 1992; 1: 53-62.

Extraction and isolation of mRNA from adult articular cartilage

We have developed a method to isolate RNA in high yield from adult articular cartilage. Homogenization of the articular cartilage with a freezer mill, extraction with 4 m guanidinium isothiocyanate/acid-phenol, and ultracentrifugation in cesium trifluoroacetate was found to be an effective and practical method for isolating a high yield of intact RNA from adult canine articular cartilage. The total RNA was suitable for Northern blot analysis. The mRNA that could then be isolated by oligo-dT affinity chromatography was found to be a suitable substrate for in vitro translation, for making a cDNA library, and for PCR amplification.