Differential expression and functionality of TRPA1 protein genetic variants in conditions of thermal stimulation.

The role of genetic modifications of the TRPA1 receptor has been well documented in inflammatory and neuropathic pain. We recently reported that the E179K variant of TRPA1 appears to be crucial for the generation of paradoxical heat sensation in pain patients. Here, we describe the consequences of the single amino acid exchange at position 179 in the ankyrin repeat 4 of human TRPA1. TRPA1 wild type Lys-179 protein expressed in HEK cells exhibited intact biochemical properties, inclusive trafficking into the plasma membrane, formation of large protein complexes, and the ability to be activated by cold. Additionally, a strong increase of Lys-179 protein expression was observed in cold (4 °C) and heat (49 °C)-treated cells. In contrast, HEK cells expressing the variant Lys-179 TRPA1 failed to get activated by cold possibly due to the loss of ability to interact with other proteins or other TRPA1 monomers during oligomerization. In conclusion, the detailed understanding of TRPA1 genetic variants might provide a fruitful strategy for future development of pain treatments.

Interleukin-1alpha treatment of meniscal explants stimulates the production and release of aggrecanase-generated, GAG-substituted aggrecan products and also the release of pre-formed, aggrecanase-generated G1 and m-calpain-generated G1-G2.

Pro-inflammatory cytokines induce meniscal matrix degradation and inhibition of endogenous repair mechanisms, but the pathogenic mechanisms behind this are mostly unknown. Therefore, we investigated details of interleukin-1 (IL-1alpha)-induced aggrecan turnover in mature meniscal tissue explants. Fibro-cartilagenous disks (3 mm diameter x 1 mm thickness) were isolated from the central, weight-bearing region of menisci from 2-year-old cattle. After 3 or 6 days of IL-1alpha-treatment, GAG loss (DMMB assay), biosynthetic activity ([(35)SO(4)]-sulfate and [(3)H]-proline incorporation), gene expression (quantitative RT-PCR) and the abundance (zymography, Western blot) of matrix-degrading enzymes and specific aggrecan products were determined. Meniscal fibrocartilage had a 4-fold lower GAG content (per wet weight) than adjacent articular cartilage, and expressed MMPs-1, -2, -3 and ADAMTS4 constitutively, whereas ADAMTS5 m-RNA was essentially undetectable. Significant IL-1 effects were a decrease in biosynthetic activity, an increase in GAG release and in the expression/abundance of MMP-2, MMP-3 and ADAMTS4. Fresh tissue contained aggrecan core protein products similar to those previously described for bovine articular cartilage of this age. IL-1 induced the release of aggrecanase-generated CS-substituted products including both high (>250 kDa) and low molecular weight (about 75 kDa) species. TIMP-3 (but not TIMP-1 and -2 or a broad spectrum MMP inhibitor) inhibited IL-1-dependent GAG loss. In addition, IL-1 induced the release of preformed pools of three known G1-bearing products. We conclude that aggrecanases are responsible for IL-1-stimulated GAG release from meniscal explants, and that IL-1 also stimulates release of G1-bearing products, by a process possibly involving hyaluronan fragmentation.

Influence of 17beta-estradiol and insulin on type II collagen and protein synthesis of articular chondrocytes.

Clinical observations have suggested a relationship between osteoarthritis and a changed estrogen metabolism in menopausal women. Type II collagen is one main structural protein of articular cartilage matrix and its synthesis is increased by insulin in growth plate cartilage. Therefore, it was investigated if [(3)H]-proline incorporation and type II collagen synthesis (immunocytochemistry, ELISA) in female bovine articular chondrocytes are affected by 17beta-estradiol and/or insulin. Articular chondrocytes were cultured in monolayers at 5% O(2) in medium containing serum for 5-9 days, followed by application of 10(-13) to 10(-9) M estradiol or 5 microg/ml insulin during a serum-free culture phase of 2-3 days. Immunostaining for type II collagen was strong in the serum-free culture phase whereas it was negative for type I collagen, indicating that cells did not dedifferentiate to fibroblast-like cells during culture in serum-free medium. Whereas insulin raised the proline incorporation and the type II collagen synthesis significantly, physiological doses of estradiol did not show significant effects. The stimulating effect of insulin on the [(3)H]-proline incorporation or the type II collagen synthesis was significantly suppressed after preincubation of cells with 10(-11) to 10(-9) M estradiol resembling an unfavorable effect for articular cartilage. The suppression was reversed if cells were incubated with 10(-11) to 10(-7) M tamoxifen or ICI 182,780 combined with 10(-11) or 10(-9) M estradiol followed by incubation with 5 microg/ml insulin, indicating an estrogen receptor-mediated process. Because the articular cartilage of diabetic patients is biomechanically less stable, further experiments are needed to clarify the role of estradiol and insulin in the metabolism of articular chondrocytes.

Pathomechanisms of cartilage destruction by mechanical injury.

Mechanical injury is considered to be a major inductor of articular cartilage destruction and therefore a risk factor for the development of secondary osteoarthritis. Mechanical injury induces damage to the tissue matrix directly or mediated by chondrocytes via expression of matrix-degrading enzymes and reduction of biosynthetic activity. As a consequence the mechanical properties of cartilage change. Some of the pathomechanisms of mechanical injury have already been uncovered by the use of a broad range of in vitro-models. They demonstrate that mechanical injury induces tissue swelling and decrease in both the compressive and shear stiffness of articular cartilage, probably due to disruption of the collagen network. Injurious compression induces chondrocyte death by necrosis and apoptosis and the remaining cells decrease their biosynthetic activity. The tissue content of proteoglycans also decreases with time in injured cartilage, and the tissue loses its ability to respond to physiological levels of mechanical stimulation with an increase in biosynthesis. Immature cartilage seems to be more vulnerable to injurious compression than more mature tissue. The expression of several matrix-degrading enzymes like ADAM-TS5 and matrix-metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9, MMP-13) is increased after injury and may in part be regulated by an autocrine vascular endothelial growth factor (VEGF)-dependent signalling pathway. Apoptosis seems to be mediated by caspase activity and reactive oxygen species. For that reason activation of antioxidative defense mechanisms as well as the inhibition of angiogenetic factors and MMPs might be key regulators in the mechanically induced destruction of cartilage and might be suggested as potential therapeutic interventions. This review summarizes some of the most important data from in vitro injury studies dealing with the pathomechanisms of cartilage destruction.

Tissue engineering of articular cartilage under the influence of collagen I/III membranes and low oxygen tension.

The objective of this study was to study the matrix production and phenotype stability of articular chondrocytes cultured on collagen I/III membranes (CM) under the influence of low oxygen tension (Po(2)). Primary bovine and osteoarthritic human chondrocytes were cultured for 2 weeks under 5-21% Po(2) on CM, in alginate, or as monolayers. Dedifferentiated cells were produced by 2-week monolayer culture under 21% Po(2). Collagen (Coll) type II and I expression was demonstrated immunohistochemically, by Western blotting (Coll II), and by semiquantitative RT-PCR; proteoglycan synthesis was demonstrated histochemically (toluidine blue); and biosynthetic activity was indicated by radiolabel incorporation ([(3)H]proline and [(35)S]sulfate). Bovine chondrocytes on CM showed an increase in Coll II expression and proteoglycan synthesis under low Po(2) conditions, whereas Coll I decreased. This oxygen-dependent phenotype-stabilizing effect was even more pronounced in alginate cultures. Biosynthesis of bovine and human chondrocytes was also increased by low Po(2), except for proline incorporation, which decreased in bovine CM cultures (low-oxygen effects were significantly higher in alginate than in CM cultures). Dedifferentiated chondrocytes reexpressed Coll II protein when cultured under low Po(2) on CM or in alginate only, but not under high Po(2) or in monolayer culture. We conclude that CM and, even more, alginate foster phenotype stability and cartilage-specific matrix production of bovine chondrocytes, especially when cultured under in vivo-like oxygen conditions.

The influence of oxygen and hydrostatic pressure on articular chondrocytes and adherent bone marrow cells in vitro.

Tissue engineering of articular cartilage from chondrocytes or stem cells is considered to be a potential aspect in the treatment of cartilage defects. In order to optimize culture conditions the influence of low oxygen tension (5%) - single or in combination with intermittent hydrostatic pressure (HP: 30/2 min on/off loading; 0.2 MPa) - on the biosynthetic activity (sulfate and proline incorporation) of human osteoarthritic chondrocytes cultured on collagen I/III membranes was investigated. Additionally, chondrogenesis from high density or monolayer cultures of bovine adherent bone marrow cells (aBMC) with and without chondrogenic medium supplements (CM) was analyzed by RT-PCR (mRNA expression of aggrecan and collagen type II). We could show that low oxygen tension increases significantly the biosynthesis of collagen I/III membrane-associated chondrocytes and even higher under co-stimulation with HP. While there is no chondrogenesis in monolayer cultures, CM induces expression of cartilage matrix molecules in high density cultures of aBMC which is even increased under the influence of low oxygen tension. Both, low oxygen tension and HP without CM are alone not sufficient stimuli for chondrogenesis. It can be concluded that low oxygen tension and HP might be useful tools in cartilage tissue engineering and that these physico-chemical factors promote but do not induce chondrogenesis under the given conditions.

The effect of estrogens and dietary calcium deficiency on the extracellular matrix of articular cartilage in Göttingen miniature pigs.

Clinical observations have suggested that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis (OA). However, positive and negative associations between the incidence of OA and serum estrogen concentrations have been reported. In contrast to this, osteoporosis is regarded as a disease with a strong estrogen-dependent component. Moreover, there is an interaction between estrogen and calcium deficiency: calcium supplementation potentiates the effect of estrogen therapy. The present study was designed to investigate how estrogen deficiency affects the articular cartilage depending on calcium supply. The distribution of different types of glycosaminoglycans and collagens can be used as an indicator for extracellular matrix changes induced by estrogen deficiency. Different levels of dietary calcium were therefore fed to intact and ovariectomized Göttingen miniature pigs for one year before articular cartilage was harvested. The histochemical staining for heavy sulfated glycosaminoglycans in the extracellular matrix of ovariectomized miniature pigs, especially of those fed with a low calcium diet, was stronger in comparison to intact animals. In intact animals type II-collagen was immunodetected in all zones of unmineralized and mineralized articular cartilage, while immunostaining for this protein was negative to weak in the deep radiated fiber zone of ovariectomized minipigs. These results suggest that the synthesis of heavy sulfated glycosaminoglycans and immunohistochemically detectable type II-collagen is possibly influenced by estrogen deficiency. In conclusion, under estrogen deficiency, the extracellular matrix of articular cartilage underwent similar changes to those observed in physiologically aging cartilage where keratan sulfate is increased as a heavy sulfated glycosaminoglycan.

Transcatheter valve implantation: damage to the human aorta after valved stent delivery system exposure--an in vitro study.

Transcatheter heart valve implantation can be performed transapically and transfemorally. The transfemoral way to the aortic valve is significantly longer than the transapical one. The aim of this study was to analyse the intima of 15 human aortas after the deployment of different conventional valved stent delivery systems. Fifteen human aortas have been analysed (77 ± 8.4 years). These aortas were preserved with formalin and explanted from the common iliac arteries to the ascending aorta. After protocolling all relevant vascular parameters, the deployment force of different conventional valved stent delivery systems was analysed. After that, the intima was closely investigated by endoscopy. The deployment force of the old catheter was not different from the actual system. The endoscopic investigation revealed significant intimal damages in all parts of the aorta after deployment of the delivery system. This study demonstrated that the passage to the aortic valve can result in significant intimal damage regardless of the used deployment catheter. Efforts are necessary to lower the profile of the deployment devices to increase their flexibility. The intima of the aorta and possible damage have to get back into the physicians' focus to avoid possible late aortic complications.

Percutaneous aortic valve replacement: emerging tractability for sufficient intracardiac resection of the aortic valve.

OBJECTIVE: : The feasibility of endovascular resection of highly calcified aortic valves has already been demonstrated by our group. Different endovascular and intracardiac tractability methods were applied. In this study, these technologies were analyzed comparing the tractability, the resection time, and the lesions in the surrounding tissue. METHODS: : All aortic valve resections (seven human hearts and 21 porcine hearts) were performed using a Thulium:YAG laser (continuous wave, wavelength of 2.01 µm, 20 watts power rating). In the first resection system, the laser fiber was controlled by a free in-lying flexible endoscope (Ø 2.5 mm, length of 600 mm). The distal part of the endoscope (40 mm) was moved in one plane by proximal manual control (three degrees of freedom). The resection system was separated into defined rooms assigning one room for one tool. The fiber was controlled by the above-mentioned endoscope (*) (three degrees of freedom). The third resection system was a mechanical microactuator carrying the laser fiber (three degrees of freedom). The fourth resection system contains a rotatable inlay with defined rooms and a newly designed nitinol (NiTi) microactuator that controlled the laser fiber (four degrees of freedom). The resection time per leaflet was measured in minutes. Gross anatomy and histology in the surrounding tissue were evaluated. RESULTS: : The resection time in approaches 1, 2, 3, and 4 was 5.5 ± 2.3 minutes, 7.4 ± 2.7 minutes, ± 6.6 minutes, and2.3 ± 1.2 minutes, respectively. The gross anatomy and histology of collateral damages revealed only superficial lesions of the surrounding tissue. The amount of lesions and the resection time were lower in the fourth approach with four degrees of freedom. CONCLUSIONS: : This analysis demonstrated that a precise tractability with four degrees of freedom is necessary for a faster and safer endovascular resection of the aortic valve. The analysis will help to optimize the ongoing development of the endovascular and intracardiac resection technology.

Percutaneous aortic valve replacement: computed tomography scan after valved stent implantation in human cadaver hearts.

Computed tomography scans were performed before and after aortic valve resection with consecutive implantation of a valved stent in human hearts with highly calcified aortic valves in situ (n=2). This demonstrates that the valved stent shows better fitting in the annulus after removal of the native valve.

Tumor necrosis factor alpha-dependent aggrecan cleavage and release of glycosaminoglycans in the meniscus is mediated by nitrous oxide-independent aggrecanase activity in vitro.

INTRODUCTION: Little is known about factors that induce meniscus damage. Since joint inflammation appears to be a causative factor for meniscal destruction, we investigated the influence of tumor necrosis factor (TNFalpha) on glycosaminoglycan (GAG) release and aggrecan cleavage in an in vitro model. METHODS: Meniscal explant disks (3 mm diameter x 1 mm thickness) were isolated from 2-year-old cattle. After 3 days of TNFalpha-treatment GAG release (DMMB assay), biosynthetic activity (sulfate incorporation), nitric oxide (NO) production (Griess assay), gene expression of matrix-degrading enzymes (quantitative RT-PCR, zymography), and immunostaining of the aggrecan fragment NITEGE were determined. RESULTS: TNFalpha induced release of GAG as well as production of NO in a dose-dependent manner, while sulfate incorporation was decreased. TNFalpha increased matrix metalloproteinase (MMP)-3 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 mRNA expression, whereas collagen type I was decreased, and aggrecan, collagen type II as well as MMP-1, -2, -13 and ADAMTS-5 were variably affected. Zymography also showed a TNFalpha-dependent increase in MMP-3 expression, but pre-dominantly in the pro-form. TNFalpha-dependent formation of the aggrecanase-specific aggrecan neoepitope NITEGE was induced. Tissue inhibitor of metalloproteinases (TIMP)-3, but not TIMP-1 or -2 inhibited TNFalpha-dependent GAG release and NITEGE production, whereas inhibition of TNFalpha-dependent NO generation with the NO-synthetase inhibitor L-NMMA failed to inhibit GAG release and NITEGE production. CONCLUSIONS: Our study shows that aggrecanase activity (a) is responsible for early TNFalpha-dependent aggrecan cleavage and GAG release in the meniscus and (b) might be involved in meniscal degeneration. Additionally, the meniscus is a TNFalpha-dependent source for MMP-3. However, the TNFalpha-dependent NO production seems not to be involved in release of proteoglycans under the given circumstances.

Percutaneous aortic valve replacement: gross anatomy and histological findings after transapical and endoluminal resection of human aortic valves in situ.

OBJECTIVE: Transluminal resection of the aortic valve was already successfully carried out by our group. The aim of this study was the analysis of the gross anatomy and the histology of the surrounding tissue after resection. METHODS: Aortic valve resection was performed in postmortem human hearts (endoluminal (EL) n=9, transapical (TA) n=4). After deployment of the aortic valve isolation chamber, the leaflets were resected with a Thullium:YAG laser scalpel (cw, 20W). After resection, the hearts were analyzed to check for lesions caused by resectioning the associated tools. Therefore, gross anatomy and histological analysis were performed (H&E staining). RESULTS: Lesions of the aortic annulus were seen in 3/9 (EL) (depth: 583+/-186 microm) and 2/4 (TA) (120 microm and one complete perforation), lesions of the aorta (ascending-arch-descending) in 4-9-0/9 (EL) and 0-0-0/4 (TA), lesions of the mitral valve in 0/9 (EL) and 0/4 (TA), lesions of the papillary muscle in 0/9 (EL) and 2/4 (TA) (depth: 400 microm and 450 microm), lesions of the endomyocardium in 0/9 (EL) and 4/4 (TA) (depth: 258+/-102 microm). The coronary ostia remained unaffected. CONCLUSIONS: This study shows fewer severe lesions in the aorta after transapical antegrade access compared to the transluminal retrograde approach. Especially noteworthy is that the aortic arch remains unaffected by the transapical procedure. These data demonstrate the transapical approach as less hazardous.

Percutaneous aortic valve replacement: endovascular sealing studies in situ.

OBJECTIVE: : To improve the outcome of percutaneous valve replacement in aortic stenosis endovascular resection of calcified aortic valves will be necessary. In this study different sealing methods were evaluated. The focus of this research was feasibility and mechanical functionality in human anatomy. METHODS: : The aortic valve isolation chamber (AVIC) is a catheter-based system to seal the aortic valve during resection, and was installed antegrade and retrograde. Firstly, AVIC was inserted antegrade via the cardiac apex in human postmortem models (n = 2), and secondly in porcine in vivo models under extra corporeal circulation (n = 5). Endoscopic inspection of the valve was recorded. AVIC was installed via a port system through the descending aorta. Micro- and macropathologies were performed. RESULTS: : AVIC transapical deployment in the two human models took 3 and 4 minutes respectively and 2.2 ± 1.3 minutes in average in the porcine model. From the descending aorta, the deployment took 9.3 ± 5.5 minutes. Fluoroscopy and macroscopy demonstrated sealed chambers. Microscopic and histologic analysis demonstrated no profound damages of the surrounding tissue. CONCLUSION: : This study demonstrates the feasibility of transapical and retrograde endovascular sealing of the aortic valve in vitro and in vivo in nonbeating hearts.

The phytoestrogens daidzein and genistein enhance the insulin-stimulated sulfate uptake in articular chondrocytes.

Clinical observations have suggested a relationship between osteoarthritis and a changed estrogen metabolism in menopausal women. Phytoestrogens have been shown to ameliorate various menopausal symptoms. Proteoglycans (PG) consisting of low and high sulfated glycosaminoglycans (GAG) are the main components of articular cartilage matrix, and their synthesis is increased by insulin in growth plate cartilage. We have investigated whether GAG synthesis and sodium [35S]sulfate incorporation in female bovine articular chondrocytes are affected by daidzein, genistein, and/or insulin. For comparative purposes, estradiol incubations were performed. Articular chondrocytes were cultured in monolayers at 5% O2 and 5% CO2 in medium containing serum for 7 days followed by the addition of 10(-11) M-10(-4) M daidzein, genistein, 17beta-estradiol, or 5 microg/ml insulin in a serum-free culture phase of 2 days. Photometrically analyzed GAG synthesis was significantly suppressed by high doses (10(-5) M-10(-4) M) of daidzein, genistein, and 17beta-estradiol. Although insulin raised the sodium [35S]sulfate uptake significantly, different concentrations of daidzein, genistein, or 17beta-estradiol showed no significant effects. However, the stimulating effect of insulin on sulfate incorporation was enhanced significantly after preincubation of cells with 10(-11) M-10(-5) M daidzein or 10(-9) M-10(-5) M genistein but not by 17beta-estradiol. In view of the risks of long-term estrogen replacement therapy, further experiments should clarify the potential benefit of phytoestrogens and insulin in articular cartilage metabolism.

Percutaneous aortic valve replacement: endovascular resection of human aortic valves in situ.

OBJECTIVE: Transluminal in vitro resection of severely calcified human aortic valves has already been successfully carried out by our group. The aim of this study was to analyze endovascular laser-assisted resection of human aortic valves in situ in 10 human cadavers. MATERIAL AND METHODS: After anterolateral minithoracotomy, the aortic valve isolation chamber system was inserted into the descending aorta and pushed forward transluminally into the aortic position to generate a separate operation space between the subvalvular and the proximal ascending aortic area. After deployment and sealing of the chamber, stable function with a continuous chamber lavage of 1.58 L/min saline solution was established (8/10 cases). The endoscopically guided laser fiber was delivered via the right carotid artery. After fixation of a leaflet by a forceps catheter, the native leaflets were resected each by a thulium:YAG laser with 20-W power rating. Macropathology and micropathology of surrounding anatomic structures were analyzed. RESULTS: The duration of transluminal positioning and deployment of the aortic valve isolation chamber took 7.3 +/- 5.8 minutes. Fluoroscopy confirmed sealed chambers. The resection was completed in all leaflets and took, on average, 6.0 +/- 3.5 minutes per leaflet. The aortic wall was moderately injured in 4 of 10 cases and the aortic annulus in two cases with one aortic wall perforation. The surrounding tissue, the coronary ostia, the mitral valve, and the left ventricular outflow tract remained unaffected. CONCLUSION: This study demonstrates the feasibility of endovascular resection of human aortic valves in situ. This is a subsequent step toward complete percutaneous replacement (resection and implantation) of human aortic valves.

Extracellular matrix changes in knee joint cartilage following bone-active drug treatment.

Certain drugs or treatments that are known to affect bone quality or integrity might have side effects on the extracellular matrix of articular cartilage. We investigated the effects of vitamin D and calcium deficiency, estrogen deficiency, and hypercortisolism alone or in combination with bisphosphonates or sodium fluoride in an animal model, viz., the Göttingen miniature pig (n=29). The articular cartilage from knee joints was analyzed for its content of glycosaminoglycans (GAGs, as macromolecules responsible for the elasticity of articular cartilage) by a spectrometric method with dimethylene blue chloride. In cryo- or paraffin sections, alkaline phosphatase (AP, as an enzyme indicating mineralization or reorganization of articular cartilage matrix) was localized by enzyme histochemistry, and positive cells were counted, whereas differently sulfated GAGs were stained histochemically. A significant decrease in GAG content was measured in ovariectomized and long-term glucocorticoid-treated animals compared with untreated animals. In the glucocorticoid/sodium fluoride group, GAGs were significantly diminished, and significantly fewer AP-positive chondrocytes were counted compared with the control. GAG content was slightly higher, and significantly more AP-positive chondrocytes were counted in short-term glucocorticoid-treated animals then in the control group. GAGs, as part of proteoglycans, are responsible for the water-storage capacity that gives articular cartilage its unique property of elasticity. Thus, ovariectomy and long-term glucocorticoid therapy, especially when combined with sodium fluoride, have detrimental effects on this tissue.

Estradiol protects cultured articular chondrocytes from oxygen-radical-induced damage.

Osteoarthritis (OA) is aggravated in menopausal women possibly because of changed serum estrogen levels. Estradiol has been postulated to affect oxidative stress induced by reactive oxygen species (ROS) in articular chondrocytes. We generated ROS in cultured bovine articular chondrocytes by incubating them with combined Fe2SO4, vitamin C, and hydrogen peroxide. The release of thiobarbituric-acid-reactive substances (TBARS, lipid peroxidation) and lactate dehydrogenase (LDH, membrane damage) was measured photometrically. Various estradiol doses and vitamin E, serving as control with an established anti-oxidative capacity, were applied either upon each exchange of medium and during radical production (strategy 1) or only during radical production (strategy 2). In chondrocytes incubated according to strategy 1, the production of TBARS and LDH release were significantly suppressed by 10(-10)-10(-4) M estradiol or by vitamin E. Under strategy 2, the production of TBARS was significantly suppressed at estradiol concentrations higher than 10(-6) M, whereas LDH release was inhibited at concentrations of 10(-6)-10(-4) M. Vitamin E showed no significant effects. As repeated application of estradiol and vitamin E produced the best results, estradiol, like vitamin E, was speculated to accumulate in the plasma membrane and to decrease membrane fluidity resulting in protection against lipid peroxidation (non-genomic effect). Thus, in contrast to the neuroprotective effect of 17beta-estradiol in supraphysiological doses reported recently, the anti-oxidative potential of estradiol appears to protect articular chondrocytes from ROS-induced damage when the hormone is given repeatedly in a physiological range. Decreased estradiol levels may therefore contribute to menopausal OA in the long term.

Influence of tissue maturation and antioxidants on the apoptotic response of articular cartilage after injurious compression.

OBJECTIVE: To study the influence of tissue maturation and antioxidants on apoptosis in bovine articular cartilage induced by injurious compression. METHODS: Bovine articular cartilage disks were obtained from the femoropatellar groove of animals ages 0.5-23 months and placed in culture. Cartilage disks were preincubated overnight with the cell-permeable superoxide dismutase (SOD) mimetic Mn(III) porphyrin (0-12.5 microM) or alpha-tocopherol (0-50 microM) and then injured by a single unconfined compression to a final strain of 50% at a velocity of 1 mm/second. After 4 days of additional incubation, the disks were fixed and embedded for light and electron microscopy. Apoptotic cells were quantified morphologically by the appearance of nuclear blebbing on light microscopy. Biosynthetic activity was demonstrated by incorporation of radiolabeled proline. The antioxidative action of the SOD mimetic was confirmed by histologic examination of cartilage after incubation with nitroblue tetrazolium. RESULTS: Injurious compression induced significantly more apoptosis in cartilage disks from newborn calves (22% of cells) than in cartilage from more mature cows (2-6%). In cartilage from 22-month-old animals, the SOD mimetic reduced the percentage of apoptotic cells induced by injury in a dose-dependent manner (complete inhibition with 2.5 microM), while alpha-tocopherol had no effect. Neither antioxidant altered protein biosynthesis or cellular ultrastructure. CONCLUSION: Our data suggest that the apoptotic response of articular cartilage to mechanical injury is affected by maturation and is mediated in part by reactive oxygen species. The antioxidative status of the tissue might be important for the prevention of mechanically induced cell death in articular cartilage.