Staining Of Cartilage Research Articles

Increased type II deiodinase protein in OA-affected cartilage and allelic imbalance of OA risk polymorphism rs225014 at DIO2 in human OA joint tissues

ObjectiveGenetic variation at the type II deiodinase (D2) gene (DIO2) was previously identified as osteoarthritis (OA) risk factor. To investigate mechanisms possibly underlying this association, we assessed D2 protein in...

Effect of functional shift of the mandible on lubrication of the temporomandibular joint

Lubrication of synovial joints reduces the coefficient of friction of the articular cartilage surface. To investigate the effect of malocclusion on the lubrication of the temporomandibular joint (TMJ), we evaluated lubricin expression in the rat TMJ immunohistochemically, under conditions of functional lateral shift of the mandible, during period of growth. Thirty 5-week-old male Wistar rats were divided into experimental, recovery, and control groups. Each rt in the experimental and recovery groups was fitted with an acrylic-plate guiding appliance. The rats in the experimental and control groups were killed at 14 and 28 days after the appliance was attached. Each rat in the recovery group was detached from the appliance at 14 days, and was killed 14 days after the appliance was removed. In the experimental group, the expression of lubricin staining in TMJ cartilage was significantly decreased during the experimental period. In the recovery group, the expression of lubricin staining in TMJ cartilage was significantly greater than in the experimental group, and there was no significant difference at 28 days between the control and recovery groups. Analysis of these data suggests that a functional lateral shift of the mandible during the growth period influences lubrication of the TMJ.

Pitx2 Deficiency Results in Abnormal Ocular and Craniofacial Development in Zebrafish

Human PITX2 mutations are associated with Axenfeld-Rieger syndrome, an autosomal-dominant developmental disorder that involves ocular anterior segment defects, dental hypoplasia, craniofacial dysmorphism and umbilical abnormalities. Characterization of the PITX2 pathway and identification of the mechanisms underlying the anomalies associated with PITX2 deficiency is important for better understanding of normal development and disease; studies of pitx2 function in animal models can facilitate these analyses. A knockdown of pitx2 in zebrafish was generated using a morpholino that targeted all known alternative transcripts of the pitx2 gene; morphant embryos generated with the pitx2ex4/5 splicing-blocking oligomer produced abnormal transcripts predicted to encode truncated pitx2 proteins lacking the third (recognition) helix of the DNA-binding homeodomain. The morphological phenotype of pitx2ex4/5 morphants included small head and eyes, jaw abnormalities and pericardial edema; lethality was observed at ∼6–8-dpf. Cartilage staining revealed a reduction in size and an abnormal shape/position of the elements of the mandibular and hyoid pharyngeal arches; the ceratobranchial arches were also decreased in size. Histological and marker analyses of the misshapen eyes of the pitx2ex4/5 morphants identified anterior segment dysgenesis and disordered hyaloid vasculature. In summary, we demonstrate that pitx2 is essential for proper eye and craniofacial development in zebrafish and, therefore, that PITX2/pitx2 function is conserved in vertebrates.

Increased type II collagen cleavage by cathepsin K and collagenase activities with aging and osteoarthritis in human articular cartilage

IntroductionThe intra-helical cleavage of type II collagen by proteases, including collagenases and cathepsin K, is increased with aging and osteoarthritis (OA) in cartilage as determined by immunochemical assays. The distinct sites of collagen cleavage generated by collagenases and cathepsin K in healthy and OA human femoral condylar cartilages were identified and compared.MethodsFixed frozen cartilage sections were examined immunohistochemically, using antibodies that react with the collagenase-generated cleavage neoepitopes, C2C and C1,2C, and the primary cleavage neoepitope (C2K) generated in type II collagen by the action of cathepsin K and possibly by other proteases, but not by any collagenases studied to date.ResultsIn most cases, the staining patterns for collagen cleavage were similar for all three epitopes: weak to moderate mainly pericellular staining in non-OA cartilage from younger individuals and stronger, more widespread staining in aging and OA cartilages that often extended from the superficial to the mid/deep zone of the tissue. In very degenerate OA specimens, with significant disruption of the articular surface, staining was distributed throughout most of the cartilage matrix.ConclusionsCleavage of collagen by proteases usually arises pericellularly around chondrocytes at and near the articular surface, subsequently becoming more intense and extending progressively deeper into the cartilage with aging and OA. The close correspondence between the distributions of these products suggests that both collagenases and cathepsin K, and other proteases that may generate this distinct cathepsin K cleavage site, are usually active in the same sites in the degradation of type II collagen.

Transinfection of rabbit knee osteoarthritis models via chitosan microsphere as gene carriers with recombined human IL-IRa gene and TGF-β1 gene

Objective To explore the method and effect of transinfection of rabbit early knee osteoarthritis models via chitosan microsphere with gene of recombined human IL-1Ra gene and TGF-β1 gene. Methods Chitosan microspheres with plasmids of IL-1Ra gene and TGF-β1 gene, and rabbit early knee osteoarthritis models were prepared. Rabbits in different groups had intra-articular injections of chitosan microsphere containing IL-1Ra gene and / or TGF-β1 gene, and chitosan solution as control group before being executed regularly and randomly. The joint specimens were evaluated by HE staining, lycopene red O staining and immunohistochemical analysis and Mankin's score. ELISA was used for detection of IL-IRa and TGF-β1 concentration of articular cavity fluid in each group. Results The control group was consistent with the pathological changes of early OA. In co-transinfection group, judging from the appearance and staining of cartilage,the OA damage of the specimens was less serious than other groups'. Its Mankin's score was significantly lower than single-gene transinfection group (P ＜ 0.05), and the latters Mankin's score were significantly lower than control group (P ＜ 0.05). Conclusion Intra-articular injection of chitosan microspheres containing both IL-1Ra gene and TGF-β1 gene could inhibit the degeneration of cartilage and promote cartilage repair. Key words: Chitosan; Interleukin-1 receptor antagonist; Transforming growth factor β1; Gene transinfection; Osteoarthritis

T-2 Toxin Induces Degenerative Articular Changes in Rodents

Osteoarthritis (OA) is a degenerative joint disease that is characterized by joint pain and a progressive loss of articular cartilage. Kaschin-Beck Disease is a form of endemic OA in China whose etiology is unclear, but epidemiological data indicate a possible link to trichothecenes mycotoxin exposure. In vitro, T-2 toxin, a trichothecenes mycotoxin, has been demonstrated to inhibit aggrecan synthesis and promote aggrecanase and pro-inflammatory cytokine production in cultured chondrocytes. To assess the effects of T-2 toxin on articular cartilage in vivo, Wistar rats were fed a diet containing T-2 toxin (100 ng/kg chow) for six and ten months. Following six months of T-2 toxin exposure, histopathological changes in femorotibial cartilage were characterized by chondrocyte degeneration/necrosis and loss, chondrocyte clones, and loss of proteoglycan staining of articular cartilage, sometimes involving the entire thickness of the cartilage in the tibial plateaus and femoral condyles. By ten months, in addition to these changes, there was evidence of cartilage fibration in some rats. In conclusion, T-2 toxin exposure in rats induced degenerative lesions in articular cartilage similar to spontaneous OA, lending support to an etiologic role of mycotoxins in Kaschin-Beck Disease. T-2 toxin-induced degenerative joint disease may be a useful model of metabolic polyarticular OA.

Morphology of pelvic fin loss in a zebrafish strain (Danio rerio)

We describe the morphology of a zebrafish strain which lacks pelvic fins but no other abnormalities. This description is the first step of analyzing hindlimb loss in an established model organism. By combining light microscopy, bone and cartilage staining, scanning electron microscopy and histological sections we were able to comprehensively describe the morphology of the developing pelvic fins of a pelvic finless zebrafish in contrast with the developing pelvic fins of wild-type zebrafish. We have shown that although adult pelvic finless zebrafish completely lack pelvic fins, they do develop mesenchymal bulges in the pelvic regions at the pelvic fin development stage. Understanding the morphology and the subsequent genetic analysis of this fish will lead to important insights into both pelvic fin/hindlimb developmental mechanisms and the evolution of hindlimb loss. It is for this reason that we present a morphological analysis of pelvic fin development and loss in this genetically tractable model species.

Determining the Proportions of Bone and Cartilage Growth in the Crucian Carp (carassius auratus) Using the Modified Simultaneous Differential Staining Technique

The modified simultaneous differential staining technique, which enables double staining of cartilage and bones, needs to be improved to prevent soft tissues from being damaged during the staining process. Key factors influencing the extent to which soft tissues are damaged include the fixative used, macerating time, potassium hydroxide concentration, incubation temperature and the removal of skin from specimens. Here we describe a protocol that enables the hardening of tissues during bleaching and maceration. We also describe a method for objectively measuring rates of cartilage and bone growth. The use of formalin as a fixative rendered soft tissues more rigid due to the resulting chemical bonds formed between proteins. Blotted specimens were immersed in 1% potassium hydroxide (KOH) and incubated at <TEX>$37^{\circ}C$</TEX> for 1 day (smaller specimens) or 2-3 days (larger specimens). The 1% KOH solution was also used as the diluent solution for the subsequent immersion in a graded series of 30%, 50%, 70%, 90%, 100% glycerol solutions, a procedure that made soft tissues even more transparent and hardened. It was not necessary to remove the skin of specimens shorter than 2 cm, since the macerating solution could easily penetrate their thin skin layer and continuously remove those pigments hindering visibility. Since excessive osmosis is another factor that can damage soft tissues in the macerating process by causing the rupture of those cells not able to withstand the osmotic pressure, here it was minimized by balancing the salt concentration between the interior and exterior of cells with the addition of 0.9% sodium chloride (NaCl) in the macerating solution. Finally, to determine the proportions of cartilage and bone growth, photographs of the stained specimens were taken with a dissecting microscope and sections corresponding to the cartilage and bones were cut out from the printed pictures and weighed. Our results show that this method is suitable for the objective evaluation of bone and cartilage growth.

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.

Restoration of Dlk1 and Rtl1 Is Necessary but Insufficient to Rescue Lethality in Intergenic Differentially Methylated Region (IG-DMR)-deficient Mice

In the Dlk1-Dio3 imprinted domain, an intergenic differentially methylated region (IG-DMR) regulates the parental allele-specific expression of imprinted genes. The maternally inherited deletion of IG-DMR (IG-DMR((-/+))) results in perinatal lethality because of the overexpression of paternally expressed genes and repression of maternally expressed noncoding RNAs (ncRNAs), including Gtl2. To better understand the possible contribution of paternally expressed genes to the lethality, we attempted to rescue the lethality of IG-DMR((-/+)) mutants by restoring the paternally expressed genes. Because the paternally inherited Gtl2 deletion (Gtl2((+/-))) induced a decrease in the expression of paternally expressed genes, we crossed female IG-DMR heterozygous mice and male Gtl2 heterozygous mutant mice. The resultant IG-DMR((-/+))/Gtl2((+/-)) double mutant mice had normal expression levels of paternally expressed genes, and none of them showed perinatal lethality; however, most mice showed postnatal lethality with decreased expression of the maternally expressed ncRNAs. Thus, we inferred that paternally expressed genes are necessary for perinatal survivability and that maternally expressed ncRNAs are involved in postnatal lethality.

Embryonic Expression of Cyclooxygenase-2 Causes Malformations in Axial Skeleton

Cyclooxygenases (COXs) have important functions in various physiological and pathological processes. COX-2 expression is highly induced by a variety of stimuli and is observed during certain periods of embryonic development. In this report, the direct effect of COX-2 expression on embryonic development is examined in a novel COX-2 transgenic mouse model that ubiquitously expresses human COX-2 from the early stages of embryonic development. COX-2 transgenic fetuses exhibit severe skeletal malformations and die shortly after birth. Skeletal malformations are localized along the entire vertebral column and rib cage and are linked to defective formation of cartilage anlagen. The cartilage anlagen of axial skeleton fail to properly develop in transgenic embryos because of impaired precartilaginous sclerotomal condensation, which results from the reduction of cell number in the sclerotome. Despite the ubiquitous expression of COX-2, the number of apoptotic cells is highly increased in the sclerotome of transgenic embryos but not in other tissues, suggesting that it is a tissue-specific response. Therefore, the loss of sclerotomal cells due to an increased apoptosis is probably responsible for axial skeletal malformations in transgenic fetuses. In addition, the sclerotomal accumulation of p53 protein is observed in transgenic embryos, suggesting that COX-2 may induce apoptosis via the up-regulation of p53. Our results demonstrate that the aberrant COX-2 signaling during embryonic development is teratogenic and suggest a possible association of COX-2 with fetal malformations of unknown etiology.

Antagonists of retinoic acid and BMP4 affect fetal mouse osteogenesis and odontoblast differentiation

Retinoic acid and bone morphogenetic protein (BMP4) are endogenous factors indispensable for the physiological development of vertebrates. The proximate aim of the present study was to investigate whether the natural compound citral (a retinoic acid synthesis inhibitor) and a monoclonal, anti-BMP4 antibody, administered to pregnant mice affect in the fetuses cranial osteogenesis and odontoblast differentiation. The present investigation was motivated by the fact that, retinoic acid inhibitors and BMP4 neutralizers may frequently contact human tissues (both intentional and unintentional, and/or unconsciously) inducing unanticipated effects. Our ultimate goal is the prevention of side effects and, future clinical implementation of the results. To this end, pregnant, white mice (balb-c Mus musculus) were intra-abdominally injected with either citral or anti-BMP4 antibody at the 9th gestational day. Newborns were processed within 5 h, postnatal. Results were evaluated (a) macroscopically, (b) stereoscopically, following histochemical double staining of cartilage and osseous tissues and, (c) microscopically after (c 1) histological staining of paraffin sections, and, (c 2) immunohistochemical detection of apoptosis. Data indicate that in vivo administration of citral (biomimicking hypovitaminosis A) caused restriction/retardation of cranial chondrogenesis and osteogenesis. Apoptosis was not detected in teeth tissues. In vivo administration of anti-BMP4 antibody resulted in a transitory interference with the normal course of odontoblast differentiation and the production of pre-dentin, whereas, delay in the ossification also included the alveoli. Animals inspected in adulthood displayed a fairly normal phenotype. It is concluded that those two substances, under their concentrations experienced, are quite safe for the public.

Xenopus development from late gastrulation to feeding tadpole in simulated microgravity

Microgravity (microG) is known to influence cytoskeletal structure, but its effects on cell migration are not well understood. To examine the effects of altered gravity on neural crest cell (NCC) migration, we inserted Xenopus laevis embryos into two separate microG-simulating slow turning lateral vessels (STLVs) just before neurulation (stage 11-12), and exposed them until feeding stage (stage 45), when the jaws and branchial apparatus are fully functional. To evaluate apparatus-related artifacts, we used two different STLVs and a vibration control as well as a stationary control vessel. Larval growth, pattern of NCC-derived cartilage formation, and incidence of malformations were analyzed using immunolocalization and wholemount staining of cartilage with Alcian blue. Interestingly, the two STLVs often yielded different or conflicting results. Many differences, such as increased cartilage size, attenuated Hoxa2 expression, and increased cell division, may be attributed mainly to vibration of the rotating vessels. However, tadpoles that developed in simulated microgravity (both STLVs, but not the vibration control) showed significantly more skeletal abnormalities, with stronger effects on cartilages derived from NCCs than those derived mainly from mesoderm. We conclude that migrating NCCs of Xenopus are sensitive to the altered gravitational environment of STLVs, and that studies relying on bioreactors to simulate microgravity also need to take variation in apparatus into account.

Development of the viscerocranial skeleton during embryogenesis of the sea lamprey,Petromyzon Marinus

Evolution of the skeleton was a key transition in early vertebrates. Lampreys lack a mineralized skeleton but possess cartilaginous neurocranial and viscerocranial elements. In lampreys, the visceral skeleton develops as a fused branchial basket supporting the pharynx. Here, we have adapted Alcian blue staining of lamprey cartilage and show this method results in cartilage fluorescence that we used to describe development of the branchial skeleton in Petromyzon marinus between 17 and 63 days of development. We show that skeletal rods develop from condensations of flattened discoidal chondrocytes and may involve cellular intercalation. Lamprey trabecular, parachordal, and subchordal cartilages consist of aggregations of polygonal chondrocytes positioned on the ventral and lateral surfaces of the notochord. We speculate that morphological differences relate to functional differences in the cartilage. We show that differentiated skeletal rods are derived from neural crest. Finally, we show how branchial muscles intercalate with skeletal rods of the branchial basket.

Enhanced Tissue Integration During Cartilage Repair In Vitro Can Be Achieved by Inhibiting Chondrocyte Death at the Wound Edge

Experimental wounding of articular cartilage results in cell death at the lesion edge. The objective of this study was to investigate whether inhibition of this cell death results in enhanced integrative cartilage repair. Bovine articular cartilage discs (6 mm) were incubated in media containing inhibitors of necrosis (Necrostatin-1, Nec-1) or apoptosis (Z-VAD-FMK, ZVF) before cutting a 3 mm inner core. This core was left in situ to create disc/ring composites, cultured for up to 6 weeks with the inhibitors, and analyzed for cell death, sulfated glycosaminoglycan release, and tissue integration. Creating the disc/ring composites resulted in a significant increase in necrosis. ZVF significantly reduced necrosis and apoptosis at the wound edge. Nec-1 reduced necrosis. Both inhibitors reduced the level of wound-induced sulfated glycosaminoglycan loss. Toluidine blue staining and electron microscopy of cartilage revealed significant integration of the wound edges in disc/ring composites treated with ZVF. Nec-1 improved integration, but to a lesser extent. Push-out testing revealed that ZVF increased adhesive strength compared to control composites. This study shows that treatment of articular cartilage with cell death inhibitors during wound repair increases the number of viable cells at the wound edge, prevents matrix loss, and results in a significant improvement in cartilage-cartilage integration.

Zebrafish Embryology and Cartilage Staining Protocols for High School Students

The Life Sciences-Howard Hughes Medical Institute Outreach Program at Harvard University supports high school science education by offering an on-campus program for students and their teachers to participate in investigative, hands-on laboratory sessions. The outreach program has recently designed and launched a successful zebrafish embryology protocol that we present here. The main objectives of this protocol are to introduce students to zebrafish as a model research organism and to provide students with direct experience with current techniques used in embryological research. The content of the lab is designed to generate discussions on embryology, genetics, fertilization, natural selection, and animal adaptation. The protocol produces reliable results in a time-efficient manner using a minimum of reagents. The protocol presented here consists of three sections: observations of live zebrafish larvae at different developmental stages, cartilage staining of zebrafish larvae, and a mutant hunt involving identification of two zebrafish mutants (nacre and chokh). Here, we describe the protocol, show the results obtained for each section, and suggest possible alternatives for different lab settings.

Vertebral deformity in cultured Atlantic cod larvae: ontogeny and effects on mortality

The effects of different egg incubation densities on the incidences of vertebral deformities in Atlantic cod larvae were investigated. Cod eggs were incubated at four different densities, 3, 6, 12 and 48 mL eggs L−1, of water. When all the eggs hatched, larvae were reared in 30 L glass aquaria. Larval samples were taken at 0, 14, 42 and 56 days post hatch (dph) for deformity analysis. Larval samples were stained using bone and cartilage staining methods to determine vertebral deformity. Incubation densities did not have any significant effects on vertebral deformities in Atlantic cod larvae. However, the incidence of larval vertebral deformity was high at hatch and decreased as the larvae grew older until 42 dph, indicating selective mortality of deformed larvae during this period. Larvae at 56 dph, however, showed an increase in the incidence of vertebral deformity, indicating a possible nutritional or prey-type effect. To our knowledge, no studies have documented the occurrence of variable patterns in vertebral deformities in cod at various developmental stages. Overall, our results suggest that broodstock husbandry, genetics and/or nutrition could play a major role in causing vertebral deformities in Atlantic cod at hatch; however, nutrition and prey type may play a major role during metamorphosis.

Collection of Embryos from Darwin’s Finches (Thraupidae, Passeriformes)

INTRODUCTIONThere are no breeding colonies of Darwin’s finches anywhere in the world. Thus, all of the embryonic material is collected in the wild. This protocol describes how, in a field setting, fertilized eggs are collected and incubated at a precise temperature and how the resulting embryos are harvested and processed for in situ hybridization, antibody staining, and microarray analyses. In addition, the protocol includes steps for preparing the heads of older embryos for histological staining of bone and cartilage with alcian blue and alizarin red. It is likely that the same or similar methods can be used to obtain embryonic tissue from other species of songbirds. The main limitation of this protocol is that, when used in the field without external constant electricity sources, it requires power generators that need to run on a more or less constant basis, as well as stockpiles of supplies (fuel, oil, and fresh water).

Alcian Blue/Alizarin Red Staining of Cartilage and Bone in Mouse: Figure 1.

INTRODUCTIONThe vertebrate skeleton forms by endochondral and intramembranous bone formation. During endochondral bone formation, mesenchyme condensations give rise to cartilages that are eventually replaced by bone. However, there are some permanent cartilages that do not ossify, such as the cartilage of the trachea and articular cartilage of the joints, and intramembranous bone formation occurs directly without a cartilage template. This article describes a method for simultaneously visualizing cartilage and mineralized tissues (notably bone) in the developing mouse. It makes use of alcian blue and alizarin red stains, and it is best used for later fetal, newborn, and early post-natal stages of development. Neonatal skeletons are especially well suited for this technique.

Early osteological development of the fins in the hatchery-reared red porgy,Pagrus pagrus(L. 1758)

Summary The present study was undertaken to establish the normal, healthy features of morphological structures at various developmental stages as achieved under well-defined environmental culture conditions (temperature between 16 and 21°C, salinity 36 ppt, pH around 7.6, and oxygen saturation over 95%) common in aquaculture of the species. The pectoral fin supports began to develop at 2.90 mm total length (TL), followed by those of dorsal fins at 5.5 mm TL, caudal fins at 5.6 mm TL, pelvic fins at 5.9 mm TL and anal fins at 6.0 mm TL. The pelvic fins appeared fully at 7.4 mm TL. Development of dorsal lepidotrichia was first observed at 6.9 mm TL, attaining their final number at 7.6 mm TL. The dorsal spines first appeared at 6.5 mm TL and were complete at 7.4 mm TL. The anal lepidotrichia appeared during the development phase from 6.8 to 8.6 mm TL. At 5.6 mm TL, the upward flexion of the urostyle was initiated. The caudal lepidotrichia formed within the primordial fin at 5.6 mm TL and reached the final count at 7.4 mm TL. The caudal dermatotrichia first appeared at 7.3 mm TL and all forms were observed by 15.5 mm TL. The development pattern of fin supports found in Pagrus pagrus is quite similar to that described for other Sparid species.