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Objective: To evaluate the structural and molecular changes in the extracellular matrix (ECM) during the process of intervertebral disc degeneration, using animal model., Methods: Wistar rats underwent intervertebral disc degeneration through 20-gauge needle puncture, and 360° rotation applied for 30 sec, representing the degenerated group, while control group was not submitted to this procedure. Histological parameters and expression of extracellular matrix molecules were evaluated in the 15(th) and 28(th) days after degenerative induction., Results: Fifteen days after the induction of intervertebral disc degeneration, significant changes were observed, such as reduction in the expression metalloprotease-9 (MMP9) and interleukins (IL-6 and IL-10). There was a significant increase in the expression of vascular endothelial growth factor (VEGF) and caspase-3. However, different alterations in the ECM were observed at 28 days, the level of collagen I, metalloprotease-2 (MMP2) and caspase-3 were enhanced. Furthermore, expression of heparanase isoforms (HPSE1 and HPSE2) mRNA were increased in the degenerative intervertebral disc., Conclusion: The different profiles of ECM molecules observed during the intervertebral disc degeneration suggest that molecular processes such as ECM remodeling, neovascularization, apoptosis and inflammation occur. Experimental Study.

It has been observed that parturition has a significant effect on female skeletal architecture and that age alters musculoskeletal tissues and their functions. We therefore hypothesized that multiparity affects the recovery of the pubic symphysis in senescent mice at postpartum and the morphology of the interpubic tissues. The pubic symphysis of primiparous young, virgin senescent (VS) and multiparous senescent (MS) Swiss mice was examined by light microscopy, transmission electron microscopy, morphometric analysis and immunohistochemistry. The mouse pubic symphysis was remodeled during the first pregnancy: the cellular phenotype and morphology changed to ensure a structurally safe birth canal, followed by recovery of the interpubic articulation after birth. The morphology of the pubic symphysis in the VS group was maintained in a state similar to that observed in virgin young mice. In contrast, MS mice exhibited an interpubic ligament characterized by extended fibrocyte-like cells, an opened interpubic articulation gap, compacted and thin collagen fibrils and scarce galectin-3-positive cells. Thus, we found that the cellular and extracellular characteristics of the pubic symphysis were altered by multiparity in senescent mice. These particular tissue characteristics of the MS group might be associated with an impaired recovery process at postpartum. Thus, a better understanding of the alterations that occur in the birth canal, including the pubic symphysis, due to multiparity in reproductively aged mice may contribute to our comprehension of the biological mechanisms that modify the skeleton and pelvic ligaments and even play a role in the murine model of pelvic organ prolapse.

Mandibular condyle articular cartilage participates in condylar postnatal growth and is responsible for adaptations to anatomical and/or biomechanical alterations throughout life. In a preliminary study in rabbits, differences were observed in the thickness of the layers of articular cartilage in control animals at 5 and 6 months (generally considered adults for this purpose). This study aimed to describe sagittally sectioned condylar cartilages stained with Picrosirius-hematoxylin in rabbits at 40 days and 5, 6, 8, 13, and 18 months to determine when histological maturity is reached. At 40 days, 5 layers were seen: fibrous, proliferative, transition, maturation, and hypertrophic. Older animals (5-18 months) lacked the transition layer. Fibrous, proliferative, and hypertrophic regions were considered for morphometric analysis. The thickness of the fibrous region did not change during the analyzed period (p = 0.1899). When proliferative and hypertrophic regions and the total thickness of the cartilage were compared, a difference was detected (p < 0.001). The thickness of the proliferative region was greatest at 40 days and decreased at 5 months; however, it increased at 6 months, when it was significantly thicker than at 5, 8, 13, and 18 months. Both the hypertrophic region and the total thickness were thickest at 40 days, intermediate at 5, 6, and 8 months, and thinnest at 13 and 18 months. In summary, our data suggest a physiological period of increased cartilage growth at 6 months. Additionally, rabbits at this age should be avoided in experiments involving condylar cartilage. Finally, 13-month-old rabbits have reached histological maturity of the condylar cartilage., (Copyright © 2011 S. Karger AG, Basel.)

Remodeling and relaxation of the mouse pubic symphysis (PS) are central events in parturition. The involvement of endogenous proteins such as matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs), and cathepsins in these phenomena remains unclear. In this work, we used a combination of immunolocalization, protein expression/activity, and relative messenger RNA (mRNA) expression to examine the changes in selected MMPs (-2, -9, and -8), TIMPs (-1 and -2), and cathepsins (B and K) during pregnancy and postpartum in mice. Immunohistochemistry revealed the presence of all of these proteins in the cytoplasm of chondrocytes, fibrochondrocytes, and fibroblast-like cells in the interpubic tissues. Zymography showed increases in the active forms of MMP-2 and -9 primarily on days 15 to 19 of pregnancy. Western blotting showed enhanced expression of MMP-8 on days 12 to 15 of pregnancy, with no changes in cathepsins B and K. Matrix metalloproteinases 2, TIMP-1 and -2, and cathepsin B had significant relative gene expression throughout pregnancy. These findings indicate that during pregnancy and postpartum there are variations in the expression and activity of proteins that may have an important role in remodeling the pubic symphysis during these events.

The aim of this study was to evaluate the effect of the pulsed ultrasound therapy (PUT) in stimulating myoregeneration and collagen deposition in an experimental model of lacerative gastrocnemius muscle lesion in 30 Wistar rats. Fifteen rats were treated (TG) daily with 1 MHz pulsed ultrasound (50%) at 0.57 W/cm(2) for 5 min, and 15 were control animals (CG). Muscle samples were analyzed on postoperative days 4, 7 and 14 through H&E, Picrosirius-polarization and immunohistochemistry for desmin. The lesions presented similar inflammatory responses in both treated and control groups. The areal fraction of fibrillar collagen was larger in the TG at 4 days post-operatively (17.53+/-6.2% vs 6.79+/-1.3%, p=0.0491), 7 days (31.07+/-7.45% vs 12.57+/-3.6%, p=0.0021) and 14 days (30.39+/-7.3% vs 19.13+/-3.51%, p=0.0118); the areal fraction of myoblasts and myotubes was larger in the TG at 14 days after surgery (41.66+/-2.97% vs 34.83+/-3.08%, p=0.025). Our data suggest that the PUT increases the differentiation of muscular lineage cells, what would favor tissue regeneration. On the other hand, it is also suggested that there is a larger deposition of collagenous fibers, what could mean worse functional performance. However, the percentage of fibers seems to have stabilized at day 7 in TG and kept increasing in CG. Furthermore, the collagen supramolecular organization achieved by the TG is also significant according to the Sirius red staining results.

The present work quantifies hyaluronan (HA) during the late pregnancy and post-partum in order to provide a better understanding of the role of HA in the adaptations that occur in the pubic symphysis during this period. HA was quantified in situ (histochemically) and in interpubic tissue extracts by fluorimetric assay. Samples were taken from virgin mice and from pregnant animals at various stages of pregnancy: 12th-18th days into pregnancy, the day of delivery (D19) and the 3rd and 5th day post-partum. The quantitative fluorimetric analysis indicated a gradual increase of HA in the interpubic tissue throughout late pregnancy (2.4-14.6 microg/mg dry weight). This was followed by a decrease beginning on D19 (12.4 microg/mg), reaching close to virgin levels (2.2 microg/mg) on the 5th day post-partum. The same optical density changes could be seen in the HA staining. Furthermore, the histochemical analysis demonstrated the presence of HA both in the extracellular matrix of the tissue and within its cells. Such results indicate that the extracellular presence of HA may contribute to the transformation of the symphysis into a flexible structure. In addition, HA's intracellular presence (until the 18th day of pregnancy) may contribute to cellular proliferation. Finally, during parturition and on the 5th day post-partum, HA may contribute to the maintenance of the myofibroblastic phenotype of ligament cells, aiding the ligament involution after parturition.

In some animals, such as mice and guinea pigs, a hormonally controlled mechanism increases the flexibility of the pubic symphysis and enhances the cervical remodeling necessary for safe delivery. Cervical ripening during pregnancy is associated with a paradoxical influx of leukocytes. However, the changes in cell metabolism during relaxation of the mouse pubic symphysis for delivery have not been extensively studied. In this work, we used light microscopy and transmission and scanning electron microcopy, as well as immunohistochemistry and Western blotting for MMP-8, to investigate the involvement of granulocytes or resident stromal cells in the relaxation of the virgin pubic symphysis during late pregnancy (days 18 and 19, before delivery) in vivo and in explanted joints. MMP-8 was studied because this collagenase is a hallmark for cervical ripening associated with the influx of granulocytes during late pregnancy. Extensive dissolution and disorganization of the extracellular matrix was seen around fibroblastic-like cells in late pregnancy. In contrast to the cervix (positive control), morphological and immunohistochemical analyses revealed that there was no characteristic cellular inflammatory response in the interpubic tissue. Staining for MMP-8 was observed in chondroid and fibroblastic-like cells of virgin and relaxed interpubic ligament, respectively. However, no granulocytes were seen during the extensive remodeling of the pubic joint in late pregnancy. These results indicate that constitutive stromal cells may have an important role in tissue relaxation during remodeling of the pubic symphysis in pregnancy., ((c) 2007 Wiley-Liss, Inc.)

Marked changes in mice pubic symphysis occur by the end of pregnancy. Tissue remodeling involves a dynamic balance between cell proliferation and programmed cell death as well as changes in the extracellular matrix components. Therefore, it is important to consider both of these cellular behaviors when investigating the mechanism that regulates interpubic tissue remodeling, growth during late pregnancy and partus ensuring involution during the postpartum period. Proliferating and programmed death cells were identified by immunohistochemistry (proliferating cell nuclear antigen and TUNEL detection, respectively) and the rates at which these processes occurred were determined by morphometric analysis. The results demonstrated that cellular proliferation was intense during the period of ligament formation, from D15 to D18, thereafter abruptly declining on D19. From parturition (D19) onwards, an ever-increasing decline in the cellular proliferation levels could be observed. The quantitative analyses of cellular death showed opposite results when compared to cellular proliferation. During early pregnancy the cycle of cellular renovation was clearly proliferative and during late mouse pregnancy the cycle was directed by programmed cellular death. Although the high levels of cellular death during postpartum involution could be shown by the TUNEL-positive cells, we were unable to observed picnotic nucleus at the light microscopy.

During pregnancy, an interpubic ligament is formed in the mouse pubic symphysis. In late stages, this ligament undergoes "relaxation" to allow proper delivery, which is expected on the 19th day. Proteoglycans and hyaluronic acid play an important role in the remodeling of the extracellular matrix in these tissues. Glycosaminoglycans and proteoglycans were studied by electron microscopic, immunohistochemical and biochemical methods in samples of mouse pubic symphysis from the 12th to 18th day of pregnancy. At the ultrastructural level, using cuprolinic blue and enzymatic digestion by chondroitin lyases, two types of proteoglycan filaments were observed in the fibrocartilage on the 12th day, as well as in D 15, D 17 and D 18 pubic ligaments. The only sulfated glycosaminoglycan in these filaments was chondroitin sulfate, as shown by chondroitin lyase treatment. Their electrophoretic mobility, before and after enzymatic degradation, corroborated this inference. The ratio of chondroitin sulfate/dry weight of symphysis showed two phases of increase: between D12 and D 15, and between D 17 and D 18. We suggest that the first corresponds mainly to an increase in decorin when the ligament is formed, and the second to versican, during "relaxation". Versican and hyaluronic acid, working as water holding molecules would be responsible for the hydration of the ligament at the end of pregnancy, allowing an increase in resiliency. The presence of hyaluronic acid was confirmed by labeling with HA-probe in the perichondrium, fibrocartilage and ligament. The role of collagen fibers as physical restrictors of the complete expansion of glycosaminoglycans and hyaluronic acid in tissue is discussed.

In many species, the cartilaginous pubic symphysis of the pregnant female is gradually replaced by a fibrous connective tissue, forming a flexible and elastic interpubic ligament. This newly formed ligament is responsible for the separation of the pubic bones, enabling safe delivery of the young. Following labor, the ligament undergoes rapid involution. To our knowledge, no previous work has focused on the phenotypic modulation that is responsible for the changes present at the interpubic ligament throughout the relaxation and closing of the symphysis. The purpose of this study was to investigate the ultrastructural features and immunophenotype of the peculiar cell type found in the pubic symphysis of cycling, pregnant and postpartum mice. In particular, immunohistochemistry studies were conducted on the expressions of the cytoskeletal proteins desmin, vimentin and alpha-smooth muscle actin (alpha-SMA). During pregnancy, the pubic symphysis cells always expressed alpha-SMA, whereas the expression of vimentin and desmin was transient from early pregnancy to postpartum. Furthermore, the expression patterns of these three cytoskeletal proteins were distinct. Cells present in the medial region of the mouse symphysis in cycling and at D12 displayed ultrastructural features characteristic of a typical fibroblast. In contrast, during the last week of pregnancy and in postpartum these cells acquired ultrastructural features representative of a myofibroblast; for example, a fibronexus and a contractile apparatus were found to be present lying in close contact with the extracellular collagenous and elastic system fibrils. Taken together, these results strongly suggest a contractile function for these cells which might contribute to support of the varying mechanical stresses present during pubic bone movement.

Reference is usually made to the parallel orientation towards the main line of exerted tension at the pubic joint in mice, for supporting forces applied to the joint. Despite the wealth of morphological information about the extracellular matrix in this joint, little is known regarding the involvement of the crimp of collagen fibers in the dramatic transformations occurring in this region during the last 3 days of pregnancy. Examination of the collagenous architecture suggests that the biomechanical properties are directly related to fibril diameters, composition of ground substance and changes in the bundle morphology, particularly in the crimp structure. The purpose of this study was to further describe the transformation of the collagen fibers of the pubic symphysis during late mouse pregnancy. We examined the architecture of collagen fibers in the symphysis and pubic ligament through the Picrosirius-polarization method and also through scanning electron microscopy to directly visualize and measure the crimping from pregnant and virgin mice. The crimp angle and the length of five consecutive crimps were measured according to Patterson-Kane et al. [Connect. Tissue Res. 36 (1997) 253]. It could be demonstrated that the angles progressively decreased and the crimp length increased, denoting that the fibers have untwisted during the relaxation process. Our findings suggest that a disruption of the helical arrangement of the collagen containing fibers may contribute to explaining the rapid remodeling that occurs at the end of pregnancy and that is responsible for an increase in pliancy and length of the pubic ligament in mice.

Proteoglycans were accurately localized in mouse pubic symphyseal tissues using the cuprolinic blue method. Specific glycosaminoglycans degradative enzymes, together with chondroitin sulfate and decorin antibodies, allowed the identification of glycosaminoglycans. Chondroitin sulfate proteoglycans were the main proteoglycans observed in hyaline cartilage, fibrocartilage, and dense connective tissue. Ultrastructurally, they were seen as electron-dense granules and filaments. The granules, rich in chondroitin sulfate chains, were exclusively found in hyaline cartilage, whereas filaments were present in cartilage, fibrocartilage, and dense connective tissue. The latter were classified by size and susceptibility to enzyme digestion into F1, F2 and F3 filaments: F1 filaments were small, thin, and collagen fibril-associated; F2 filaments were thick, heavily stained, and localized around individual collagen fibrils and between bundles of collagen fibrils; and F3 filaments were scattered throughout elastic fiber surfaces. Considering their localization, susceptibility to chondroitinase AC and immunohistochemical detection, the symphysial F1 filaments were found to be preferentially decorin substituted with chondroitin sulfate side chains. The F2 filaments were also susceptible to chondroitinase AC treatment, whereas F3 filaments could be digested by heparitinase. The data thus obtained on the localization and identification of pubic symphyseal proteoglycans in virgin mice may be useful in the study of structural modifications that occur throughout pregnancy.

We described the behaviour of 120 days rabbit knee-meniscus cells in monolayer culture. The cells were grown forming cellular aggregates resembling true cellular nodules. Three stages of development of these nodules could be observed: formation of the cellular nodules between days 1 and 3; nodular growth, with their maximal at day 5; and nodular regression beginning at day 8. Ultrastructural analysis of the extracellular matrix of these cellular nodules was assessed on days 3, 5 and 8. At the formation stage, we could observe striated collagen fibrils and small bundles of tubular microfibrils either interspersed with very low quantities of amorphous elastin, being morphologically identical to elaunin fibers, or without only trace of elastin, being morphologically identical to oxytalan fibers. By day 5, fibrillar elements with 100 nm periodic ladder-like collagen VI fibrillar aggregates could also be detected. At day 8, the striated collagen fibrils and oxytalan fibers could not be observed. During this same period, there was an increase of a dense matrix comprised of collagen VI and mature elastic fibers. Chondroitin/dermatan sulfate proteoglycans were synthesized and became essential for the arrangement of collagen type VI, since chondroitinase ABC treatment of the culture disrupted collagen VI assembly, associated with the large spaces near the cell surface. In addition, the cells lost their fusiform morphology and changed into rounded cells. The results show that primary cultures of rabbit meniscus fibrochondrocytes maintain their capacity to form chondro-like structures in vitro. The organization process was rapid and uniform throughout the entire culture presuming that the normal signal transduction pathways are maintained intact and that essential factors in some phases of tissue organization are present.

Current literature concerning smooth muscle blood vessels has shown versican as the main proteoglycan (PG) component of the matrix. To show whether smooth muscle matrix has the same PG distribution when present in organs, other than the blood vessels, the inner circular smooth muscle layer of the small intestine was obtained by dissection as a highly purified tissue and analyzed by biochemical and cytochemical methods. The smooth muscle layer PGs were extracted from dog small intestine with 4 M guanidine-HCl in the presence of proteinase inhibitors, purified by charge equilibrium, isolated by equilibrium CsCl density gradients, and analyzed in terms of anion exchange, size, and glycosaminoglycan (GAG) distribution. Proteoheparan sulfate itself represented 91.5% of the PGs present in this tissue. The remainder was proteodermatan sulfate. Cytochemical analyses using the cationic dye cuprolinic blue associated with enzymatic treatments with chondroitinases ABC and heparitinase III showed the arrangement and identification of PGs in basal lamina and intramuscular connective tissues. The PGs in the basal lamina were proteoheparan sulfate, and those associated with collagen fibrils in the endomysium and perimysium were rich in dermatan sulfate. In contrast to the blood vessels, inner circular muscle smooth tissue in intestine has, as the main PG, perlecan.

The present study was designed to assess the influence of aqueous and nonaqueous fixatives on the quantitative estimation of collagen-proteoglycan interaction in tissue sections. Tissues containing different collagen types and distinct sulfated proteoglycan classes were isolated from pig costal cartilage, human skin, and the inner muscular layer of dog small intestine and fixed using aqueous or nonaqueous methods. The results showed that the best fixation method was exposure to paraformaldehyde gas. When using aqueous fixatives, proteoglycans were lost to different degrees among the various tissues analyzed, reflecting differences in chemical properties of proteoglycan classes and/or in their interactions with other matrix components such as collagen.

Glycosaminoglycan composition of normal saphenous veins and atherosclerotic saphenous vein grafts is reported. Dermatan sulfate is the main glycosaminoglycan present in both normal saphenous veins and saphenous vein grafts. These tissues also contain chondroitin sulfate and heparan sulfate. Although the total amount of glycosaminoglycans decreased in the grafts (compared with normal saphenous veins), the grafts showed an increase in the relative amounts of dermatan sulfate and chondroitin sulfate. Heparan sulfate was decreased, compared with normal controls. These findings suggest the involvement of blood vessel glycosaminoglycans (not only the arterial glycosaminoglycans) in the process of atherosclerosis.

A major proteoglycan (PG) in rabbit meniscal fibrocartilage was extracted with 4 M guanidine-HCl. It represented 63% of the total uronic acid originally present in the tissue. The non-extractable PG could be released by treating the guanidine HCl-extracted tissue with trypsin followed by alkaline hydrolysis. The glycosaminoglycan (GAG) chains from the non-extractable PGs are shown to be of a different type than the extractable forms since the relative amounts of chondroitin sulphate isomers differ from each other.

In spite of the fact that the various anatomical regions of a given articular cartilage surface are subjected to different degrees of stress, the present observations strongly suggest that there exists a topographical homogeneity in the distribution of glycosaminoglycans in the same articular cartilage. In contrast to this age-related changes in the proportion of the different types of glycosaminoglycan species in articular cartilage are remarkable. Non-sulphated chondroitin could only be detected in very young articular cartilage. Dermatan sulphate, which has already been detected in young adult rabbits, was followed by the appearance of keratan sulphate in older rabbits. Chondroitin 4-6-sulphates were detected in all articular cartilages studied, the proportion of the 6-sulphated variably increasing with age. The present report suggests that the distribution of glycosaminoglycans in articular cartilage varies with species and age, and the data can further vary, depending on the methods used. It is therefore concluded that generalizations against the results reported in the literature should be considered skeptically.

A simple method was used to isolate hyaluronic acid (HA) and sulfated glycosaminoglycans from synovial fluids obtained from children with juvenile rheumatoid arthritis (JRA) and from normal age-matched controls. The bulk of the glycosaminoglycans present in both normal and pathologic synovial fluids consisted of hyaluronic acid and chondroitin 6-sulfate. The diseased synovial fluids showed a sharp decrease in the concentration of glycosaminoglycans when compared with normal controls. These findings are similar to those reported for the synovial fluid of adult patients with rheumatoid arthritis.

The following basement membranes (BMs) from representative species of the main vertebrate classes were studied by the Picrosirius-polarization method: lens capsule, Reichert's membrane and glomerular BMs. A distinct birefringence was consistently observed in all BMs from all species studied by this method. The results reported provide a strong evidence for collagen macromolecular orientation in BMs. Heparitin sulphate was the only glycosaminoglycan detected in dogs lens capsules.

Corneal explants with scleral rims were freshly prepared from day 18 chicken embryos and incubated in vitro for 3 h in the presence of various radioactive precursors. Radiolabeled proteoglycans were isolated from the stromal tissue and culture medium for analysis. Two predominant proteoglycans were identified in corneal stroma. One contains dermatan sulfate and the other contains keratan sulfate; a structural analysis of each is reported in the accompanying paper (Midura, R.J., and Hascall, V.C. (1989) J. Biol. Chem. 264, 1423-1430). A minor keratan sulfate proteoglycan distinct from the major form, a small amount of heparan sulfate proteoglycan, and some sulfated glycoproteins were also detected in stromal extracts. The biosynthesis of the dermatan sulfate proteoglycan was stable in vitro and in ovo, whereas that of the major keratan sulfate proteoglycan was stable only in ovo. Various treatments were tried to maintain a high rate of keratan sulfate synthesis with time in culture. Cooling the corneal explants to 5 degrees C was the only treatment that reduced this decline in keratan sulfate synthesis in vitro to any significant extent. Three major proteoglycans were observed in the culture medium. Two were dermatan sulfate proteoglycan and appeared to be mainly derived from the scleral tissue surrounding the corneal explant. The third proteoglycan contained keratan sulfate. It was smaller in size and lower in charge density compared to the keratan sulfate proteoglycan found in the stroma, but both appeared to have similar core protein sizes. It seems likely that this proteoglycan was synthesized in the stroma and secreted into the medium. A small amount of heparan sulfate proteoglycan and some sulfated glycoproteins were also detected in the medium.

Biopsy specimens of nonpregnant and intrapartum human cervices were studied by electron microscopy, and by the Picrosirius-polarization method which is a specific procedure for the detection of collagen fibers in tissue sections. The results obtained demonstrate a marked reduction of collagen fibers in the intrapartum material as compared to the samples of nonpregnant cervices. Evidence that indicated the presence of a widespread collagenolysis in the intrapartum biopsy specimens was obtained by both optical and electron microscopy, thus suggesting that this might be the main cause of the changes in the cervix that permit it to dilate during parturition. The study of the neutrophilic polymorphonuclear leukocytes in the intrapartum biopsy specimens strongly suggested that these cells participate in this process.

A qualitative and quantitative biochemical study of the glycosaminoglycans was performed in tissues constituted predominantly by one type of collagen, or in tissues containing mixtures of different types of collagen. The results obtained show the presence of dermatan sulfate, chondroitin sulfate, and heparin sulfate in tissues containing collagen types I, II, or III, respectively, suggesting a specific correlation of different glycosaminoglycans with these three types of collagen.

This paper reports the existence of mast cells in an epithelial location in the gallbladders of both cattle and sheep. The histochemical studies performed on these cells showed that their cytoplasmic granules contain heparin and biogenic amines in both species. Optical- and electron microscopic observations demonstrated that, in both species, mast cells from the connective tissue of the gallbladder diapedese across the basal lamina and migrate through the epithelium all the way to the luminal surface, and that a degranulation process takes place during this migration. The biochemical results showed a correlation between the number of mast cells present in the epithelium and the amount of heparin detected in the different regions of the gallbladders of the species studied. Unusually high contents of heparin were found in both cattle and sheep gallbladders, suggesting that they should be studied as possible commercial sources of this polimer.

Differences in the glycosaminoglycans and proteoglycans synthesized by "young," "adult," and tumoral chondrocytes are reported. Young cartilage and human chondrosarcoma contain chondroitin 4- and 6-sulfates, whereas adult human cartilage contains almost exclusively chondroitin 6-sulfate. High keratan sulfate content is reported in adult cartilage, whereas it is almost absent in young and tumoral cartilages. The electrophoretic pattern and keratan sulfate content in these proteoglycans from adult cartilage are clearly distinct from those of the young and tumoral cartilages. The high molecular weight is the distinguishing property of the glycosaminoglycan synthesized by tumoral chondrocytes.

The sulfated glycosaminoglycan content of primary cultures of fibroblasts from guinea-pig embryo kidney is reported. A hybrid chondroitin sulfate comprises approx. 90% of these glycosaminoglycans from the cell coat. Changes in the proportion of labelled heparitin sulfate were also observed after successive subcultures. We postulate a possible correlation between the pattern of glycosaminoglycans and processes of cell selection and cell dedifferentiation in these cultures.

Ultrastructural and histochemical studies performed on the skeletal elements of the tail fins of six representative species of teleosts enabled the following observations to be made. The electron microscopic pattern of amorphous substance deposition, and the diameter of the collagen fibrils, in lepidotrichia closely resemble those which are typical of cartilage. In addition, lepidotrichia contain chondroitin sulfate AC as the only sulfated glycosaminoglycan, and this glycosaminoglycan shows high levels of interaction with collagen, both features being characteristic of cartilage. Furthermore, the histochemical data presented in this paper suggest that not all of the glycosaminoglycans present in lepidotrichia are bound to protein cores to form proteoglycans. Each actinotrichium consists of a single ultrastructural entity of remarkable width and, thus, is not composed of a bundle of discretely separated collagen fibrils but rather of hyperpolymerized collagen molecules. This aspect differs from the arrangement pattern of all the other interstitial collagens, suggesting that actinotrichia may contain a new type of collagen.

The connective tissue matrix of hepatic granulomas from Swiss mice, each infected with 20 Schistosoma mansoni cercariae, was examined using morphological, histochemical and biochemical methods, during the evolution of the disease. Evidence was obtained by both light and electron microscopy that suggested the presence of two different collagen populations which appeared successively during granuloma evolution. Morphological features suggesting the occurrence of collagenolysis were observed at the final stage of granuloma development. The connective tissue matrix of schistosome granulomas was devoid of elastic system fibres. Schistosome egg granulomas contain dermatan sulphate-proteoglycan.

A comparative study on the distribution of sulfated mucopolysaccharides in several tissues of five mammalian species is reported. It is shown that each tissue has a characteristic composition differing from each other regarding the relative amount, type and molecular size of chondroitin sulfate A/C, chondroitin sulfate B and heparan sulfate. It is also shown that the same tissue from different mammals has the same types and proportions of sulfated mucopolysaccharides, but with different molecular weights. Exception to this rule was observed for the distribution of heparin which was present only in a few tissues of the five mammals studied. The possible involvement of the sulfated mucopolysaccharides in cell recognition and/or adhesiveness is discussed in view of this characteristic distribution.

A new type of cartilage was found participating in a valve-like system inside the conus arteriosus of the fresh water sting ray, Potamotrygon sp.. This cartilage possesses no chondrocytes and its matrix is perforated by vascular channels that ramify dendritically forming canaliculi. The acellular cartilage does not possess perichondrium but, rather, it is attached to a basement membrane-like structure. The cartilaginous matrix contains collagen fibrils that strongly interact with the chondroitin sulfate of the ground substance. The histochemical and biochemical findings suggest that not all of the glycosaminoglycans present in the acellular cartilage are bound to protein cores to form proteoglycans.

The guinea pig mesentery is a uniform, continuous, thin (18 micron) sheet of connective tissue covered by a single layer of flattened mesothelial cells on both surfaces. Tight and gap junctions provide for cell-to-cell adhesion among mesothelial cells. These cells possess numerous micropinocytotic vesicles; a conspicuous basal lamina separates the mesothelium from the underlying connective tissue. Most of the material found between the two serous coverings consisted of a three-dimensional meshwork of abundant collagenous fibers intermingled with a sparse net of very thin (0.4 micron) elastic fibers. Two distinct populations of collagen fibrils are segregated into different compartments of the mesentery. One population is formed of thick (56 nm) fibrils which associate to form closely packed fibers. The second population, composed of loosely arranged thin (38 nm) fibrils which do not become assembled into fibers, is found underlying the basal lamina that separates the mesothelium from the connective tissue. These observations strongly suggest that the mesentery contains both collagens type I and type III. The guinea pig mesentery contains 6.8 mg of sulfated glycosaminoglycans/g dry weight. Most of these glycosaminoglycans (78%) were identified as dermatan sulfate, whilst the rest (22%) corresponded to heparan sulfate.

This paper reports the existence of granulated cells in the bovine gallbladder epithelium. The histochemical studies performed on these cells showed that their cytoplasmic granules contain heparin. Optical- and electron microscopic observations demonstrated that these mast cells migrated from the adjacent connective tissue, and crossed the basal membrane in order to enter the epithelium and reach the luminal surface.