safranine-t and Osteoarthritis

Malleolar fracture, which is present in 37-53% of human ankle osteoarthritis (OA), is the most common type of fracture in the ankle joint. In spite of this, no rat animal model has been developed for this type of injury to date. Here, we established a rat ankle post-traumatic OA (PTOA) model induced by malleolar fracture; this model will be useful in ankle OA research.. Two-month-old male Sprague Dawley (SD) rats were randomized into 2 groups (n = 19 per group): 1) malleolus articular fracture, dislocation, and immediate reduction on the right joints and 2) malleolus articular fracture on the right ankle. The contralateral ankle joints were used as controls. The fracture and healing processes were confirmed and monitored by radiography. Changes in inflammation were monitored in vivo by fluorescence molecular tomography (FMT). Cartilage damage and changes in expression of OA-related genes were analyzed by histology, immunohistochemistry, Real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) at 8 weeks post-surgery.. X-rays showed that all fractures were healed at 8 weeks post-surgery. A reproducible, mild to moderate degree of OA cartilage damage with reduced aggrecan was detected by histology in all animals in both groups but there was no significant difference between the two groups. Decreased Col-II and increased Col-X and MMP-13 levels were detected by qPCR, immunohistochemistry, ELISA and FMT from both groups cartilage.. Malleolus articular fracture alone induces ankle OA with lesions on the central weight bearing area of the tibiotalar joint in rats. This model will provide a reproducible and useful tool for researchers to study ankle OA.

Topics: Aggrecans; Animals; Ankle Fractures; Ankle Joint; Arthritis, Experimental; Biomarkers; Cartilage, Articular; Humans; Indicators and Reagents; Male; Molecular Imaging; Osteoarthritis; Phenazines; Radiography; Random Allocation; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Tomography, Optical

Transforming growth factor-beta (TGF-β) plays an important part in the repair of cartilage in osteoarthritis. It has been hypothesised that intra-articular injection of TGF-β₁ promotes repair of cartilage and protects the subchondral bone from damage in osteoarthritic temporomandibular joints (TMJs). We made bilateral partial perforations of the disc to induce osteoarthritic joints in 36 rabbits. TGF-β₁ 20, 40, or 80 ng were injected into the right joint, and vehicle alone was injected into the left joint. Four additional animals were used as normal controls. Microcomputed tomography was used to quantify the three-dimensional microarchitecture of subchondral bone, followed by assessment of the proteoglycan content. All joints treated with TGF-β₁ were covered by a layer of well-organised fibrocartilage, and had increased proteoglycan content and normal microarchitectural properties, whereas the joint treated by vehicle alone had typical osteoarthritis-related degradation of cartilage and sclerosis of subchondral bone. These results suggested that TGF-β₁ is an effective way of treating osteoarthritis of the TMJ.

Topics: Animals; Bone Density; Calcification, Physiologic; Cartilage, Articular; Chondrocytes; Coloring Agents; Disease Models, Animal; Fibrocartilage; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Injections, Intra-Articular; Male; Mandibular Condyle; Osteoarthritis; Phenazines; Proteoglycans; Rabbits; Random Allocation; Sclerosis; Temporomandibular Joint Disc; Temporomandibular Joint Disorders; Transforming Growth Factor beta1; X-Ray Microtomography

The temporomandibular joint (TMJ) is an important growth and articulation center in the craniofacial complex. In aging it develops spontaneous degenerative osteoarthritic (OA) lesions. Metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPS) play key roles in extracellular matrix remodeling and degradation. Gelatinase activities and immunohistochemical localization of MMP-2, -3, -8, -9, and -13 and TIMP-1 and -2 were examined in mandibular condyle cartilage of neonatal mice up to 18 months old. The most intense immunostaining for all enzymes and TIMPs and the peak of gelatinase activities were found in animals in the stages of early growth (1 week to 3 months) followed by a decrease during maturation and aging. However, clusters of positively immunoreactive chondrocytes were detected in cartilages of old animals displaying OA lesions. Positive safranin-O staining, indicative of sulfated proteoglycans (PGs), was prominent in the TMJ of newborn mice up to 3 months old followed by reduction during maturation and aging, except in regions displaying OA lesions. Temporal codistribution of PGs, MMPs, and TIMPs during skeletal maturation reflected an active growth phase, whereas their reduction coincided with the more quiescent articulating and maintenance phase in the joint cartilage. Osteoarthritic lesions were associated with both increased PG synthesis and MMP immunoreactivity, indicating limited repair activity during initial stages of osteoarthritis.

Topics: Aging; Animals; Cartilage; Immunohistochemistry; Indicators and Reagents; Matrix Metalloproteinases; Mice; Osteoarthritis; Phenazines; Proteoglycans; Temporomandibular Joint; Tissue Inhibitor of Metalloproteinases

Adult articular cartilage is divided by the tidemark into a deep calcified layer and a more superficial uncalcified layer. Histologic examination of articular cartilage from the knee joint of golden Syrian hamsters 123 days of age or older revealed defects at the tidemark in the tibia. Defects ranged from small separations of the calcified and uncalcified layers along the tidemark to progressively larger defects apparently formed by dissolution. These larger defects appeared as cavities in the noncalcified cartilage, had smooth rather than rough edges, frequently contained coalesced debris, and often resulted in a bulge in the articular surface. Occasionally, these large defects broke through the articular surface. Defects were not observed in tibial cartilage of younger (<90 days old) hamsters or in femoral cartilage from hamsters of any age. Exercise neither protected against nor increased the severity of the defects. Collagen cross-linking by pyridinoline was examined as a function of age and increased from 1,090 to 3,062 micromoles of pyridinoline/mole of hydroxyproline over the period of 1-9 months of age but was not correlated with defect formation. With increasing age, these focal tidemark defects could lead to osteoarthrosis-like cartilage lesions.

Topics: Age Factors; Animals; Calcinosis; Cartilage, Articular; Chromatography, Liquid; Collagen; Cricetinae; Eosine Yellowish-(YS); Female; Hematoxylin; Hindlimb; Hydroxyproline; Indicators and Reagents; Joints; Mesocricetus; Osteoarthritis; Phenazines; Physical Conditioning, Animal; Rodent Diseases

The depletion of the pericellular and territorial matrices in articular cartilage is considered to be one of the earliest events in pathobiology of osteoarthritis (OA). A newly discovered family of proteins with a disintegrin-like and metalloproteinase-like domain (ADAM) may be involved in matrix degradation as well as in cell-cell and cell-matrix interactions. The purpose of this study was to determine by in situ hybridization whether human articular chondrocytes from newborn, normal adult, and OA cartilages express messenger RNA for ADAM-10, one of the members of this family, and by semiquantitative RT-PCR to compare the levels of this expression. The results confirmed the expression of ADAM-10 by human articular chondrocytes and revealed the highest levels of expression in the continuously remodeling cartilage of newborns and the most fibrillated areas of OA cartilage, especially the regions of cell clusters. Importantly, ADAM-10 mRNA expression was evident in tissues with the greatest loss of Safranin O staining from the territorial and interterritorial matrix of the chondrocytes. Messenger RNA was upregulated in OA tissue compared to the age-matched normal cartilage, as detected by RT-PCR. Upregulated levels of ADAM-10 mRNA expression appear to be related to the degree of cartilage damage and/or degradation, which suggests a potential role for at least one member of this new family in the cartilage matrix destruction accompanying OA.

Topics: ADAM Proteins; ADAM10 Protein; Adult; Aged; Aged, 80 and over; Aging; Amyloid Precursor Protein Secretases; Cartilage, Articular; Cells, Cultured; Child, Preschool; Coloring Agents; Female; Humans; In Situ Hybridization; Infant; Infant, Newborn; Male; Membrane Proteins; Metalloendopeptidases; Middle Aged; Osteoarthritis; Phenazines; Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic

A slowly progressive osteoarthritis model in the skeletally immature canine knee joint is described. Forty-four young female beagle dogs were chosen as experimental animals. In 15 dogs, a 30 degrees valgus angulation of the right tibia was created by operation. Fourteen dogs underwent sham operation. Fifteen dogs served as control subjects. Alterations in the knee joints were evaluated macroscopically and histologically 7 and 18 months after operation. Seven months after surgery, two of seven beagles that had valgus osteotomy had a lesion with discoloration of cartilage in the medial condyle of the femur. Eighteen months after operation, five of the eight dogs that had valgus osteotomy showed fibrillation of the femoral and tibial cartilages. Mankin's scoring of the knee joint cartilages indicated statistically significant changes as compared with control subjects 7 and 18 months after surgery. Biomechanical analysis revealed shift of the mechanical axis toward the lateral compartment of the knee by the valgus osteotomy, patellofemoral malalignment, and inclination of the tibiofemoral joint line. These biomechanical alterations brought about the most severe cartilage lesions to the medial condyle of the femur and the patellofemoral joint. Cartilage fibrillation took more than 7 months to develop. Thus, this model offers a slowly progressive, well standardized, and reproducible method for the study of early changes of osteoarthritis in young beagle dogs.

Topics: Animals; Biomechanical Phenomena; Cartilage, Articular; Chondrocytes; Coloring Agents; Disease Models, Animal; Disease Progression; Dogs; Female; Femur; Follow-Up Studies; Hindlimb; Menisci, Tibial; Osteoarthritis; Osteotomy; Patella; Phenazines; Reproducibility of Results; Tibia

A method of image analysis has been developed for use in the semiquantitative histomorphometric assessment of glycosaminoglycans in articular cartilage stained with safranin O. The reliability of the methodology is reported along with its application to the assessment of articular cartilage in a model of osteoarthritis, i.e., transection of the anterior cruciate ligament in rabbits. With this system, specimens of normal and osteoarthritic articular cartilage were assessed histomorphometrically for the following parameters: total cartilage area, percentage of safranin O stained area, mean gray scale (average stain intensity), and gray scale index (the relative total amount of glycosaminoglycans). Reproducibility was established for 12 specimens of normal cartilage and found to have a SD of less than 8% of the mean for each parameter that was measured. Image analysis of osteoarthritic cartilage revealed each of the parameters, except for average stain intensity, to be significantly lower than that in control cartilage.

Topics: Animals; Cartilage, Articular; Disease Models, Animal; Femur Head; Glycosaminoglycans; Image Processing, Computer-Assisted; Indicators and Reagents; Knee Joint; Osteoarthritis; Phenazines; Rabbits; Reproducibility of Results

We studied whether cyclic loading is harmful to degraded cartilage. Sets of four cartilage-bearing sesamoid bones were dissected from 5-year old cows. One bone from each set was cultured for 17 h in control medium to serve as an ex vivo control. The three others were cultured for 1 week in control medium to which 0, 10 or 300 ng/mL retinoic acid (RAc), which depletes the cartilage matrix of proteoglycans, had been added. Two were then cultured for another week in control medium. During the last week, one of the two was subjected to a cyclic load (1 MPa, 0.2 Hz). Following treatment with RAc, glycosaminoglycan content and synthesis were significantly decreased, as confirmed by safranin O staining and autoradiography. They were further diminished by loading during the second week of culture. Increased amounts of 3-B-3(-)epitope were found in cartilage that had been treated with 300 ng/mL RAc and then loaded. While loading cartilage matrix that was only slightly degraded proved to be damaging, loading severely degraded cartilage matrix apparently induced osteoarthritic-like changes.

Topics: Animals; Autoradiography; Cartilage, Articular; Cattle; Cells, Cultured; Extracellular Matrix; Female; Glycosaminoglycans; Keratolytic Agents; Osteoarthritis; Phenazines; Stress, Mechanical; Tretinoin; Weight-Bearing

In situ hybridization and immunohistochemical techniques were applied to investigate gene expression and extracellular deposition of collagen type II in normal, osteoarthritic and rheumatoid human articular cartilage. Normal cartilage showed an essentially even extracellular distribution of type II collagen with poly- and monoclonal antibodies, while only a few cells were positive for alpha 1(II) collagen mRNA. In situ hybridization of osteoarthritic and rheumatoid cartilage, however, showed strong enhancement of type II collagen gene expression; transcripts were observed predominantly in the upper middle zone of the articular cartilage while the upper layer was mostly negative and correlated with a zone of reduced proteoglycan staining. The elevated mRNA levels frequently coincided with pericellular immunostaining for type II collagen, indicative for enhanced synthesis of the protein. In two samples, however, pericellular loss of collagen type II staining was found despite positive cytoplasmic signals with the alpha 1(II) RNA probe, suggesting enhanced collagen destruction. Control hybridization with a probe for 18S rRNA revealed very few negative cells throughout both normal and arthritic cartilage samples, ruling out major cell necrosis in the specimens investigated. Thus, our observations identify sites of activated type II collagen synthesis in osteoarthritic cartilage that were predicted by previous biochemical studies and support the notion that damaged cartilage attempts to restore matrix by enhanced synthesis of its components.

Topics: Adolescent; Adult; Aged; Arthritis, Rheumatoid; Cartilage, Articular; Collagen; Histocytochemistry; Humans; Immunohistochemistry; In Situ Hybridization; Middle Aged; Osteoarthritis; Phenazines; Proteoglycans; Reference Values; RNA Probes; Staining and Labeling; Tolonium Chloride

The intensity of safranin 'O' staining is directly proportional to the proteoglycan content in normal cartilage. Safranin 'O' has thus been used to demonstrate any changes that occur in articular disease. In this study, staining patterns obtained using monoclonal antibodies against the major components of cartilage proteoglycan chondroitin sulphate (anti CS) and keratan sulphate (anti KS), have been compared with those obtained with safranin 'O' staining, in both normal and arthritic tissues. In cartilage where safranin 'O' staining was not detectable, the monoclonal antibodies revealed the presence of both keratan and chondroitin sulphate. Thus, safranin 'O' is not a sensitive indicator of proteoglycan content in diseases where glycosaminoglaycan loss from cartilage has been severe.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Arthritis; Arthritis, Rheumatoid; Cartilage, Articular; Chondroitin Sulfates; Extracellular Matrix; Humans; Immunohistochemistry; Keratan Sulfate; Middle Aged; Osteoarthritis; Phenazines; Proteoglycans