Latexin expression correlated with mineralization of articular cartilage during progression of post-traumatic osteoarthritis in a rat model.

As latexin has been linked with chondrocyte hypertrophic differentiation it is possible that this protein may also be involved in the mineralization of cartilage in OA. Therefore, we correlated latexin expression with the mineralization marker, alkaline phosphatase and determined the mineral deposition in the articular cartilage by analyzing the Ca/P ratio and the collagen fibrils pattern, during the progression of post-traumatic OA in a rat model. OA was induced by medial meniscectomy and post-surgery exercise for 5, 10, 20 and 45 days. Protein expression in articular cartilage was evaluated by immunofluorescence, histochemistry and Western blot. Minerals and structure of collagen fibrils in the superficial zone of cartilage were analyzed by energy dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM) respectively. Protein expression analysis showed time-dependent up-regulation of latexin during OA progression. In the cartilage, latexin expression correlated with the expression and activity of alkaline phosphatase. EDX of the superficial zone of cartilage showed a Ca/P ratio closer to theoretical values for basic calcium phosphate minerals. The presence of minerals was also analyzed indirectly with AFM, as the collagen fibril pattern was less evident in the mineralized tissue. Latexin is expressed in articular cartilage from the early stages of post-traumatic OA; however, minerals were detected after latexin expression was up-regulated, indicating that its activity precedes and remains during the pathological mineralization of cartilage. Thus, our results contribute to the identification of molecules involved in the mineralization of articular chondrocytes.

Menisectomized miniature Vietnamese pigs develop articular cartilage pathology resembling osteoarthritis.

Animal models have been used to understand the basic biology of osteoarthritis (OA) and have helped to identify new candidate biomarkers for the early diagnosis and treatment of this condition. Small animals cannot sufficiently mimic human diseases; therefore, large animal models are needed. Pigs have been used as models for human diseases because they are similar to humans in terms of their anatomy, physiology and genome. Hence, we analyzed articular cartilage and synovial membrane pathology in miniature Vietnamese pigs after a unilateral partial menisectomy and 20-day exercise regimen to determine if the pigs developed pathological characteristics similar to human OA. Histological and protein expression analysis of articular cartilage from menisectomized pigs revealed the following pathologic changes resembling OA: fibrillation, fissures, chondrocyte cluster formation, decrease in proteoglycan content and upregulation of the OA-associated proteins MMP-3, MMP-13, procaspase-3 and IL-1ß. Moreover, histological analysis of synovial membrane revealed mild synovitis, characterized by hyperplasia, cell infiltration and neoangiogenesis. Pathological changes were not observed in the contralateral joints or the joints of sham-operated pigs. Further studies are required to validate such an OA model; however, our results can encourage the use of pigs to study early stages of OA physiopathology. Based on their similarities to humans, pigs may be useful for preclinical studies to identify new candidate biomarkers and novel treatments for OA.

Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis.

The Integrin ß1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differentiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-ß) Superfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedifferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressed αV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of the α5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hypertrophy.

Exercise modulates the expression of IL-1ß and IL-10 in the articular cartilage of normal and osteoarthritis-induced rats.

After a joint lesion, high-impact exercise is a risk factor for the development of osteoarthritis (OA). The degradation of articular cartilage in OA has been associated with the activation of inflammatory cytokine signaling pathways. However, differences in cytokine expression in healthy and injured cartilage after exercise have not yet been analyzed. We used immunofluorescence and Western blot to study the expression of IL-1ß and IL-10 in the articular cartilage of normal (N), sham-operated (S), and menisectomized (OA) rats subjected or not to high-impact exercise (E) for 3, 6, and 10 days (N, NE, S, SE, and OA groups). Cartilage integrity and proteoglycan content were only affected in the OA groups. Exercise increased the amount of IL-1ß and IL-10 positive chondrocytes in NE and SE groups compared with non-exercised groups (N and S). The expression of IL-1ß was up-regulated over time in the NE and OA groups, although in the late stages the increase was higher in the OA groups. In contrast, the expression of anti-inflammatory IL-10 was low in the OA group, whereas in the NE groups expression levels were higher at each time point analyzed. These results suggest that anti- and pro-inflammatory molecules in the cartilage might be tightly regulated to maintain the integrity of the tissue and that when this equilibrium is broken (when the meniscus is removed), the pro-inflammatory cytokines take over and OA develops.

Identification of latexin by a proteomic analysis in rat normal articular cartilage.

BACKGROUND: Osteoarthritis (OA) is characterized by degeneration of articular cartilage. Animal models of OA induced are a widely used tool in the study of the pathogenesis of disease. Several proteomic techniques for selective extraction of proteins have provided protein profiles of chondrocytes and secretory patterns in normal and osteoarthritic cartilage, including the discovery of new and promising biomarkers. In this proteomic analysis to study several proteins from rat normal articular cartilage, two-dimensional electrophoresis and mass spectrometry (MS) were used. Interestingly, latexin (LXN) was found. Using an immunohistochemical technique, it was possible to determine its localization within the chondrocytes from normal and osteoarthritic articular cartilage. RESULTS: In this study, 147 proteins were visualized, and 47 proteins were identified by MS. A significant proportion of proteins are involved in metabolic processes and energy (32%), as well as participating in different biological functions including structural organization (19%), signal transduction and molecular signaling (11%), redox homeostasis (9%), transcription and protein synthesis (6%), and transport (6%). The identified proteins were assigned to one or more subcellular compartments.Among the identified proteins, we found some proteins already recognized in other studies such as OA-associated proteins. Interestingly, we identified LXN, an inhibitor of mammalian carboxypeptidases, which had not been described in articular cartilage. Immunolabeling assays for LXN showed a granular distribution pattern in the cytoplasm of most chondrocytes of the middle, deep and calcified zones of normal articular cartilage as well as in subchondral bone. In osteoarthritic cartilage, LXN was observed in superficial and deep zones. CONCLUSIONS: This study provides the first proteomic analysis of normal articular cartilage of rat. We identified LXN, whose location was demonstrated by immunolabeling in the chondrocytes from the middle, deep and calcified zones of normal articular cartilage, and superficial and deep zones of osteoarthritic cartilage.

Expression of MIG-6, WNT-9A, and WNT-7B during osteoarthritis.

Although the molecular mechanisms for initiation of cartilage destruction in osteoarthritis (OA) are unknown, it has been demonstrated that disruption of mitogen-inducible gene 6 (Mig-6) in mice leads to the onset of a degenerative joint disease like OA. On this basis, we correlated gene expression of Mig-6 with Wnt-9a and Wnt-7b genes; we showed downregulation of Mig-6, Wnt-7b, and Wnt-9a during OA, while Wnt-7b was expressed also in osteoblast-like cells. Here we suggest that Aggrecan degradation occurs before the downregulation of Mig-6. It remains to be proven whether there is any relation between Wnt signaling and Aggrecan degradation.

Chondrocytes interconnecting tracks and cytoplasmic projections observed within the superficial zone of normal human articular cartilage--a transmission electron microscopy, atomic force microscopy, and two-photon excitation microscopy studies.

The morphology of the normal human and rat articular cartilage was assessed using transmission electron microscopy (TEM), atomic force microscopy (AFM), and two-photon excitation (2PE) microscopy. Spurr-embedded sections from fixed human cartilage were simultaneously evaluated using TEM and AFM. The presences of tracks among the chondrocytes from the superficial zone of the cartilage were observed. In order to ratify the presence of interconnecting tracks among superficial zone chondrocytes, whole fixed human and rat cartilage, as well as fresh whole rat cartilage, were examined under the 2PE. In all cases, these tracks were observed. In addition, porous matrix, well-defined lacunae, and cytoplasmic projections anchored to the extracellular matrix (ECM) were also detected. We conclude that normal human and rat flattened superficial chondrocytes might be interconnected by tracks running through the ECM. In addition, cytoplasmic projections were observed anchored to the ECM. All these structures may possibly be related to cell/cell and ECM/chondrocytes signaling. Our findings provide new information that possibly will be of relevant importance for a more profound study of normal cartilage physiology and eventually, the pathogenesis of osteoarthritis.

Identification of exocytotic membrane proteins, syntaxin-1 and SNAP-25, in Entamoeba histolytica from hamster liver.

Entamoeba histolytica is a protozoan parasite causing dysentery and in some cases liver abscesses. These effects have been attributed to cytolytic substances released by exocytosis. In this study, the presence of the proteins syntaxin-1 and SNAP-25, which are assumed to be involved in exocytosis, were examined by immunohistochemistry, immunoelectron microscopy and western blot analysis. Syntaxin-1 and SNAP-25 were expressed in the vesicular, vacuolar and plasma membranes of E. histolytica trophozoites. It can be concluded that these proteins might be involved in exocytosis processes.

Ontogeny of rat chondrocyte proliferation: studies in embryo, adult and osteoarthritic (OA) cartilage.

The aim of this work was to study the ontogeny of chondrocyte cell division using embryo, adult and osteoarthritic (OA) cartilage. We searched for mitosis phases and performed a comparative evaluation of mitotic index, basic fibroblast growth factor b (FGFb), transforming growth factor beta1 (TGF-beta1) receptors, cyclin dependent kinase (CDK1) and Cyclin-B expression in fetal, neonate, 3, 5, 8 weeks old rats and experimental OA. Our results showed that mitosis phases were observed in all normal cartilage studied, although, we found a decrease in mitotic index in relation to tissue development. No mitosis was detected in OA cartilage. We also found a statistical significant reduction in cell number in OA cartilage, compared with the normal tissue. Furthermore, FGFb and TGF-beta1 receptors diminished in relation to tissue development, and were very scarce in experimental OA. Western blot assays showed CDK-1 expression in all cases, including human-OA cartilage. Similar results were observed for Cyclin-B, except for 8 weeks, when it was not expressed. Our results suggest that cell division seems to be scarce, if not absent within the OA cartilage studied. Nevertheless, the existence of factors essential for cell division leaves open the question concerning chondrocyte proliferation in OA cartilage, which is likely to be present in the early stages of the disease.

The bactericidal effect of silver nanoparticles.

Nanotechnology is expected to open new avenues to fight and prevent disease using atomic scale tailoring of materials. Among the most promising nanomaterials with antibacterial properties are metallic nanoparticles, which exhibit increased chemical activity due to their large surface to volume ratios and crystallographic surface structure. The study of bactericidal nanomaterials is particularly timely considering the recent increase of new resistant strains of bacteria to the most potent antibiotics. This has promoted research in the well known activity of silver ions and silver-based compounds, including silver nanoparticles. The present work studies the effect of silver nanoparticles in the range of 1-100 nm on Gram-negative bacteria using high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM). Our results indicate that the bactericidal properties of the nanoparticles are size dependent, since the only nanoparticles that present a direct interaction with the bacteria preferentially have a diameter of approximately 1-10 nm.

A new role for chondrocytes as non-professional phagocytes. An in vitro study.

Chondrocytes are capable of engulfing latex particles, cell detritus, and necrotic and apoptotic remains in vitro. It is conceivable that chondrocytes might be involved in the clearance by phagocytosis of different materials within the cartilage. In fact, so far there is no evidence for the presence of "professional phagocytes" (macrophages and neutrophils) in this tissue. Chondrocyte suspensions obtained from rat knees and hips were cultured to assess phagocytosis of latex particles (1 microm), articular cartilage detritus, and necrotic and apoptotic chondrocyte remains (induced by VP-16 1 mM). We observed that chondrocytes phagocytosed latex particles as evaluated by confocal microscopy and flow cytometry. In addition, we observed that chondrocytes phagocytosed articular cartilage detritus and necrotic and apoptotic VP-16 induced-chondrocytes, as observed by bright field microscopy and transmission electron microscopy.

Modifications of Golgi complex in chondrocytes from osteoarthrotic (OA) rat cartilage.

The status of the Golgi complex in normal vs osteoarthrotic (OA) cartilage has not yet been studied. A monoclonal antibody, MAb 58-K-9, allowed scoring of Golgi labeling intensity. In addition, ultrastructural assessment enabled us to focus on the distribution and relation between the endoplasmic reticulum (ER) and Golgi membranes. The study was performed in both normal and partially menisectomized OA-induced rat cartilage 20 and 45 days after surgery. Comparing Golgi immunolabeling intensities (mean +/- SEM) revealed a highly significant difference between normal (9.98 +/- 1.25), 20-day (2.49 +/- 0.34), and 45-day (0.82 +/- 0.22) cartilage. Moreover, chondrocytes from normal cartilage displayed 71.18% of labeling intensity in contrast to OA cartilage, in which chondrocyte labeling intensities were 24.95% (20 days) and 8.11% (45 days). OA chondrocytes appeared to display an overall reduction in Golgi labeling intensity, suggesting disruption of this organelle as the OA damage progressed. Interestingly, many 20-day OA-induced chondrocytes exhibited bubble-like Golgi immunolabeling compartmentalizing the cytoplasm, concomitant with putative apoptotic nuclear changes. At the same time, OA chondrocytes with a typical ultrastructural apoptotic pattern revealed a prominent ER gathered together with Golgi vesicles and saccules, also appearing to compartmentalize chondrocyte cytoplasm. We speculate about the role of Golgi modifications and apoptosis in OA pathogenesis.

Preservación de córneas para trasplante en medio de cultivo MCB / Corneal preservation for transplant in MCB culture

Se compara la viabilidad endotelial de córneas de conejo, conservadas en tres medios diferentes durante 48, 72 y 96 horas valoradas mediante la tinción con azul de tripano, microscopia de luz y microscopia electrónica. Material y métodos: Fueron analizadas 20 córneas divididas de la siguiente manera: ocho se conservaron en Optisol, 10 en MCB y dos en solución salina balanceada, mantenidas a 4o C. Resultados: En las córneas mantenidas en solución salina balanceada se observó destrucción de las células endoteliales a las 48 horas. En córneas preservadas en MCB y Optisol se apreció destrucción de un 5-10 por ciento de las células a las 72 horas. La destrucción fue del 20 por ciento después de 96 horas y de 30 por ciento luego de 105 horas en las células preservadas en el medio MCB. El estudio de microscopia electrónica de transmisión también confirmó que las células estructuralmente se encuentran preservadas hasta las 90 horas tanto en Optisol como en MCB. Conclusión: El medio de preservación para córneas producido en México (MCB) mantiene el tejido corneal hasta por 90 horas, de manera similar al Optisol, pero a un costo considerablemente menor.

Variability in the cell phenotype of aggregates or "clones" of human osteoarthritic cartilage. A case report

Human samples of articular cartilage from the knee of a clinically classified osteoarthritic patient, assessed by arthroscopy as part of the surgical treatment was studied by light and transmission electron microscopy. This particular case differed from others already reported in the variability of cell phenotype within the aggregates or "clones" frequently present in the osteoarthritic cartilage. The most common morphology of "clonal" cells forming the aggregates were large and rounded with an euchromatic nucleus. The cytoplasm was characterized by the presence of alternately clear and dense sites. At the ultrastructural level it was seen that the clear sites were formed by disrupted intermediates filaments and small particles, and that the dense sites were constituted by the segregation of different organelles of the chondrocytes. In addition, there were atypical aggregates composed only by secretory cells or by degenerating chondrocytes. Furthermore, a complex structure consisting of a very large cell inside a giant lacunae delimited by electron-dense material with small vesicles is described as a novel finding. The variability in the chondrocyte phenotype of the aggregates described here could be an indication of a better prognosis; nevertheless, the follow-up of the evolution of this patient is needed in order to know the final outcome.

Estimulación de los receptores de macrofagos en bajas concentraciones iónicas de Na+, K+, Ca++ y Mg++: potenciales de transmembrana / Stimulation of macrophages receptors in low ionic concentrations of Na+, K+, Ca++, Mg++: transmembrane potentials

Las células mononucleares fagocíticas obtenidas de la cavidad peritoneal de la rata, cambian las magnitudes de los potenciales de transmembranas de acuerdo con el estímulo fagocítico y en relación con el receptor estimulado. La incubación de estas células en medios iónicos, donde indistintamente se les bajó las concentraciones extracelulares de Na+, K+, Ca++ y Mg++ mostraron despolarizaciones de la membrana antes de inducir la fagocitosis que estimularon los receptores específicos Fc con latex cubierto con inmunoglobulinas IgG en los medios bajos Na+, Ca+, y Mg++, no se modificaron para los medios de K+. El estímulo fagocítico; provocó hiperpolarizaciones de la membrana; para todos los cambios iónicos pero fueron de menor magnitud para los decrementos de Na+, Ca+, y Mg++, respecto a lo registrado en las soluciones tyrode normales. Los tiempos en que ocurren estos cambios de PT también se modifican. Estos resultados muestran la importancia de las concentraciones iónicas extracelulares en la capacidad fagocítica de los macrófagos