Cell types and evidences for traumatic cell death in a pressure-bearing tendon of Rana catesbeiana

Tendon fibrocartilages appear in areas subjected to compressive forces. The bullfrog plantaris longus tendon was shown to be subjected to compression and to develop a modified region which differs from fibrocartilage in many respects. Ultrastructural analyses of the compression region of the bullfrog tendon demonstrated the existence of typical fibroblasts in the fibrous areas and large cells with abundant cytoplasm filled with intermediate type filaments. This large cell type has organelles restricted to a small perinuclear area or dispersed in the network of intermediate type filaments. Other cells were also found and exhibited less abundant deposition of intermediate filaments, showing an organization intermediate between fibroblasts and typical cells from the compression region. These intermediate type cells are closely associated with collagen bundles while the large cells seemed to have no connection with the fibrous components, but are immersed in a glycosaminoglycan-rich extracellular matrix. Aspects of cell death in association with extracellular matrix disruption were observed in some instances and it is likely that these are associated with traumatic stimulation of the tendon, especially when it is submitted to the sudden and strong mechanical loading expected to occur during jumping. Since the damage occurred mainly in cells of the intermediate type, it is assumed that accumulating intermediate type filaments is a protective mechanism against compressive forces to which this tendon is subjected.     

The role of tenascin-C in adaptation of tendons to compressive loading

Although most tendon regions are subjected primarily to high tensile loads, selected regions, primarily those that directly contact bones that change the direction of the tendon, must withstand high compressive loads as well. Compressed tendon regions differ from regions subjected to primarily tensile loads: they have a fibrocartilaginous structure with spherical cells surrounded by a matrix containing aggrecan and collagen types I and II, in contrast regions not exposed to compression have a fibrous structure with spindle shaped fibroblasts surrounded by a matrix of dense, longitudinally oriented type I collagen fibrils. The spherical shape of cells in fibrocartilagenous regions indicates these cells are more loosely attached to the matrix than their spindle-shaped counterparts in fibrous regions, a feature that may help to minimize cell deformation during tendon compression. We hypothesized that expression of tenascin-C, an anti-adhesive protein, is part of the adaptation of tendon cells to compression that helps establish and maintain fibrocartilaginous regions. To test this hypothesis we compared tenascin-C content and expression in compressed (distal) versus uncompressed (proximal) segments of bovine flexor tendons. Immunohistochemistry and immunoblot analyses showed that tenascin-C content was increased in the distal tendon where it co-distributed with type II collagen and aggrecan. Tendon cells from the distal segments expressed more tenascin-C than did cells from the proximal segments for up to four days in cell culture, indicating that increased tenascin-C expression is a relatively stable feature of the distal cells. These observations support the hypothesis that tenascin-C expression is a cellular adaptation to compression that helps establish and maintain fibrocartilagenous regions of tendons.     

Histochemistry defines a proteoglycan-rich layer in bovine flexor tendon subjected to bending

Mid-substance fibrocartilage develops in bovine deep flexor tendon at the point where the tendon wraps under sesamoid bones of the foot and receives transverse compressive loading during locomotion. Fibrocartilage extends several millimeters into the tendon at this location and the proteoglycan-rich tissue stains intensely with Alcian blue. Using histochemical techniques we demonstrate the presence of aggrecan, type VI collagen, and hyaluronic acid in the extracellular matrix of this region of tendon. Biglycan staining was localized to the cells, however. Adjacent to the fibrocartilage, at the outer curvature of the tendon as it bends, the tissue resembles typical tensile tendon with dense bundles of linearly arranged collagen. Longitudinal sections revealed discrete layers of Alcian blue-stained material between the collagen bundles. We demonstrate that these layers of loose matrix also contain aggrecan, type VI collagen, and hyaluronic acid. However, the dense collagen bundles of this region are negative for these components. Transverse sections of tendon in the area adjacent to fibrocartilage show a distinct Alcian blue-stained structure surrounding vascular elements at the point where several fiber bundles come together. This is concluded to be the same structure as the Alcian blue-stained layers seen in longitudinal sections. These observations suggest that proteoglycan-rich matrices in tendon subjected to mechanical loading other than pure tension may serve multiple roles. Such matrices can not only provide compressive stiffness and separate and lubricate collagen bundles that move relative to each other, but may also protect the integrity of vasculature in tendon subjected to bending and shear.     

Proteoglycan synthesis by fibroblast cultures initiated from regions of adult bovine tendon subjected to different mechanical forces

Fibroblast cultures were initiated from two distinct regions of the adult bovine deep flexor tendon and synthesis of 35S-labeled proteoglycans by these cultures was investigated. The proximal/tensional region of the tendon was composed of linearly arranged dense collagen bundles, and its glycosaminoglycan hexosamine content was only 0.2% of the dry weight of the tissue. The proteoglycans of this region were predominantly small (Kav = 0.5 on Sepharose CL-4B). Cells placed into culture from this region attached to the substratum readily, and the radiolabeled proteoglycans from these cultures were 90% small proteoglycans. In a more distal region of the tendon that is subjected to compressive forces, the collagen was arranged as a network of fibrils separated from each other by a matrix that stained intensely with Alcian blue. The glycosaminoglycan content of this compressed region was up to 5-fold higher than in the proximal region, and as much as 50% of the proteoglycans were large molecules (eluted from Sepharose CL-4B in the Vo). Cells placed into culture from the distal/compressed region did not attach to the substratum as readily as those from the proximal region and were characterized by the presence of numerous cytoplasmic lipid inclusions. The [35S]proteoglycans synthesized by the distal tendon fibroblast cultures were divided into two approximately equal populations of large and small proteoglycans having elution characteristics similar to the proteoglycans extracted from this tissue. The distinct profiles of proteoglycan production were maintained by the cells in culture for several weeks, although eventually the amount of large proteoglycan synthesized by the distal tendon fibroblast cultures diminished. Both regions of tendon contained predominantly type I collagen, and collagen production was about 10% of the total protein synthesized by both cell cultures. These observations indicate that adult tendon fibroblasts in culture express stable synthesis of proteoglycan populations similar to those found in the region of tendon from which they were derived.     

Arachnoid mater of the bullfrog,Rana catesbeiana

Summary In the bullfrog, the meninges surrounding the central nervous system include an arachnoid mater that contains layers of cells with abundant intermediate filaments (IFs) having unique organizational characteristics. This membrane contains an inner lamina of cells that resemble fibroblasts and an outer lamina of flattened cells that are almost filled with IFs. The IFs of the outer arachnoid are arranged in compact, arching bundles that lie parallel to the outer surface of the central nervous system. Thus, sections cut tangentially to the membrane reveal bending of filament bundles, whereas transverse sections do not. In some cells bordering the subdural space, bundles of filaments are organized into highly-ordered spiral arrays. Attachments to the numerous desmosomes and, apparently, to the nuclear envelope suggest anchoring of cytoplasmic structures by the IF system. Microtubules occur primarily near the plasma membrane and the nucleus. Numerous caveolae also are associated with the plasma membrane.The unusual abundance, organization, and cytoplasmic relations of IFs in the bullfrog arachnoid suggest that this membrane may serve as an important model for study of fundamental cytoskeletal relations and function.     

Ultrastructure of reticulum cells in the bone marrow

In this study the attempt was made to classify the reticulum cells of the bone marrow on the basis of electron-microscopic findings. The basis of the differentiation was the ability of the cells to phagocytize substances or not. For two cell types the intracytoplasmic filaments were used as distinctive marks. The following classification resulted: (a) phagocytic reticulum cells, (b) undifferentiated reticulum cells, (c) fibrous reticulum cells of type I, which contain filaments of 4-8 nm diameter and are located near the blood sinus of the bone marrow, (d) fibrous reticulum cells of type II, which contain intracytoplasmic filaments of 10 nm diameter; since these cells contain neutral fat bodies, the possibility of a reversible conversion to fat cells has to be assumed and (e) fibroblasts, cells which synthesize the substance of the extracellular space. A connection of reticulum cells to haematopoietic functions or to stem cell functions could be found.     

The myotendinous junction of the smooth feather muscles (mm. pennati)

Summary Smooth feather muscles (mm. pennati) consist of bundles of smooth muscle cells which are attached to the feather follicles by short elastic tendons. In addition, some muscle bundles are interrupted by elastic tendons. The elastic tendon is composed of longitudinally arranged elastic fibers which branch and wavy bundles of collagen fibrils. Smooth muscle cells of the muscle bundles are attached to each other by desmosome-like junctions and by fusion of the basal laminae. The cytoplasm of the muscle cells is characterized by conspicuous thick filaments and abundant thin and intermediate filaments. These are attached to band-like dense patches (dense bands) at the plasma membrane which are particularly broad at the tapering end of the muscle cell. The contact surface between smooth muscle cells and their elastic tendon is considerably increased (i) by deep finger-like invaginations and indentations located at the tapering muscle end, and (ii) by branching of the coarse elastic fibers into slender processes, which are attached to the richly folded surface of the muscle cell endings by peripheral microfibrils. This intimate interlocking closely resembles the myotendinous junctions in skeletal muscle. In addition to fibroblasts and fibrocytes, the myotendinous junction of the young growing chicks contains numerous so-called myofibroblasts, which are suggested to represent smooth muscle cells differentiating into fibroblasts of the developing tendon.Dedicated to Professor Dr. Helmut Leonhardt on the occasion of his 60th birthdaySupported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91/1)     

Type VI collagen in extracellular, 100-nm periodic filaments and fibrils: identification by immunoelectron microscopy

Filaments and fibrils that exhibit a 100-nm axial periodicity and occur in the medium and in the deposited extracellular matrix of chicken embryo and human fibroblast cultures have been tentatively identified with type VI collagen on the basis of their similar structural characteristics (Bruns, R. R., 1984, J. Ultrastruct. Res., 89:136-145). Using indirect immunoelectron microscopy and specific monoclonal and polyclonal antibodies, we now report their positive identification with collagen VI and their distribution in fibroblast cultures and in tendon. Primary human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, showed a progressive increase in labeling and changes in distribution with time up to 8 d in culture. With immunoelectron microscopy and monoclonal antibodies to human type VI collagen followed by goat anti-mouse IgG coupled to colloidal gold, they showed in thin sections specific 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) attached to the band region and another (4B10) to the interband region of the filaments and fibrils. Rabbit antiserum to type VI collagen also localized on the band region, but the staining was less well defined. Control experiments with antibodies to fibronectin and to procollagen types I and III labeled other filaments and fibrils, but not those with a 100-nm period. Heavy metal-stained fibrils with the same periodic and structural characteristics also have been found in both adult rat tail tendon and embryonic chicken tendon subjected to prolonged incubation in culture medium or treatment with adenosine 5'-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils represent the native aggregate form of type VI collagen. It is likely that banded fibrils of the same periodicity and appearance, reported by many observers over the years in a wide range of normal and pathological tissues, are at least in part, type VI collagen.     

Relationship between connective tissue cells and fibronectin in a sequential model of experimental hepatic fibrosis

The cellular and non-cellular components of fibrous septa formed at early and late stages in a sequential model of experimental hepatic fibrosis have been investigated using ultrastructural and immunocytochemical techniques. In the early septa, cells with intermediate features between lobular Ito cells and active fibroblasts were formed. These cells frequently displayed subplasmalemmal microfilaments (myofibroblast-like cells). Macrophages were also present. Scanty typical fibroblasts were present in the late septa. This cellular recruitment might be related to an extracellular glycoprotein-fibronectin-which is at present under investigation as a chemotactic factor for fibroblasts. Strong positivity for fibronectin in early septa and its sharp decrease in late septa seems to support this view. Fibroblasts and/or macrophages are the likely source of fibronectin synthesis.     

Proteoglycan synthesis in organ cultures from regions of bovine tendon subjected to different mechanical forces.

Synthesis of proteoglycans by morphologically and chemically distinct regions of bovine flexor tendon was investigated in explant cultures. Proximal regions of the flexor tendon which experience only tensile forces and have low contents of proteoglycans initially exhibited relatively low rates of proteoglycan synthesis but high rates of collagen synthesis. The predominant proteoglycan produced by all proximal explants was of small hydrodynamic size and appeared similar to that extracted from proximal tissue. In contrast, explants derived from the distal tendon region, which experiences frictional and compressive forces in addition to tensile forces, and has a high content of proteoglycans, showed relatively high initial rates of proteoglycan synthesis and lower rates of collagen synthesis. These distal explants produced primarily large proteoglycans on the first day in culture. Turnover of newly synthesized proteoglycans was not detectable in proximal tissue, and was low in distal tissue. Loss of unlabelled proteoglycan from proximal and distal explants was not detected during the 12 days of culture. These observations suggest that the increase in specific types of proteoglycans in regions of tendon subjected to frictional and compressive forces is the result of elevated synthesis rates in this tissue. Two alterations in proteoglycan synthesis occurred during the 12-day culture period. (1) The rate of proteoglycan synthesis by all explants increased with time in culture. (2) The proportion of small proteoglycans synthesized by distal explants increased from 32% of the total proteoglycan produced on day 1, to 80% of that produced on day 12. Explants from proximal tendon continued to produce only small proteoglycans throughout the 12 days in culture. This switch in proteoglycan phenotype, resulting in decreased synthesis of large proteoglycans by the distal tissue, may be due to a lack of compressive forces on the cultured explants.     

Posttranslational regulation of keratins: degradation of mouse and human keratins 18 and 8.

Human keratin 18 (K18) and keratin 8 (K8) and their mouse homologs, Endo B and Endo A, respectively, are expressed in adult mice primarily in a variety of simple epithelial cell types in which they are normally found in equal amounts within the intermediate filament cytoskeleton. Expression of K18 alone in mouse L cells or NIH 3T3 fibroblasts from either the gene or a cDNA expression vector results in K18 protein which is degraded relatively rapidly without the formation of filaments. A K8 cDNA containing all coding sequences was isolated and expressed in mouse fibroblasts either singly or in combination with K18. Immunoprecipitation of stably transfected L cells revealed that when K8 was expressed alone, it was degraded in a fashion similar to that seen previously for K18. However, expression of K8 in fibroblasts that also expressed K18 resulted in stabilization of both K18 and K8. Immunofluorescent staining revealed typical keratin filament organization in such cells. Thus, expression of a type I and a type II keratin was found to be both necessary and sufficient for formation of keratin filaments within fibroblasts. To determine whether a similar proteolytic system responsible for the degradation of K18 in fibroblasts also exists in simple epithelial cells which normally express a type I and a type II keratin, a mutant, truncated K18 protein missing the carboxy-terminal tail domain and a conserved region of the central, alpha-helical rod domain was expressed in mouse parietal endodermal cells. This resulted in destabilization of endogenous Endo A and Endo B and inhibition of the formation of typical keratin filament structures. Therefore, cells that normally express keratins contain a proteolytic system similar to that found in experimentally manipulated fibroblasts which degrades keratin proteins not found in their normal polymerized state.     

Increased organization of cytoskeleton accompanying the aging of human fibroblasts in vitro

Fibroblastic cells of human origin have a limited lifespan in culture. One of the senescence-associated phenotypic changes is an increase in the abundance of cytoplasmic filaments. Human skin fibroblasts (strain 0011), derived from an 8-week-old male fetus, were passaged according to a predetermined schedule and examined at successive population doubling levels. In young rapidly growing cultures, fluorescence microscopy with NBD-Phallacidin shows a normal organization of the actin-containing fibers, microtubules and intermediate filaments, as has been described previously. At stages close to the end of the in vitro lifespan of the cell strain, large flat fibroblasts are the predominant cell type in culture. These large senescent fibroblasts contain numerous prominent actin fibers traversing the entire long axis of the cytoplasm. The fibers are often located adjacent to each other and appear to form a sheet on the ventral side of the cytoplasm. Staining of senescent cells with anti-tubulin antibody reveals an increase in the abundance of microtubules per cell and the distribution pattern is altered through the increase in the number of organization centers. Intermediate filaments are also more abundant and display tightly packed fibrillar sheets or bundles. Electron microscopic studies have confirmed the increased organization of microfilaments into bundles in senescent cells. These results suggest that during in vitro senescence, the increase in cell size is correlated with increased organization of the cytoskeleton. The presence of a rigid cytoskeletal structure may contribute in part to the inability of the senescent cell to replicate.     

Immunohistochemical studies on the tissue localization of collagen types I, III, IV, V and VI in schwannomas. Correlation with ultrastructural features of the extracellular matrix

The distinctive tissue localization of collagen types in typical schwannomas with Antoni type A and B areas was demonstrated immunohistochemically using affinity-purified antibodies against types I, III, IV, V and VI collagen and comparative ultrastructural studies were made on the extracellular matrix components. Antoni type A tissue, which was composed of tightly packed spindle cells with long cytoplasmic processes surrounded by a continuous basement membrane and a few fibrillar components of the extracellular matrix, was almost exclusively immunoreactive for type IV collagen, presumably representing the basement membrane. Verocay bodies, which are organoid structures of Antoni type A tissue, had a variety of more abundant extracellular fibrous components, such as banded collagen fibrils, fibrous long-spacing fibrils and microfibrils. These were positive for type I and III, as well as type IV collagen. In Antoni type B areas, where two types to tumor cells designated Schwann cell-like and fibroblast-like were scattered in large amounts of amorphous extracellular matrix containing microfibrils and thick banded collagen fibrils, type VI collagen as well as types I, III and IV collagen were consistently detected. Type V collagen was localized in dense fibrous tissue areas and around blood vessels. These findings indicate that the differently organized cellular patterns of schwannomas, identified as Antoni types A and B, are characterized not only by the ultrastructural features of the extracellular matrix, but also by the distinctive collagen types produced by neoplastic Schwann cells.     

Organization of desmin-containing intermediate filaments during differentiation of mouse decidual cells

Decidualization of the mouse endometrium consists of a redifferentiation of the endometrial stromal fibroblasts. During decidualization these fibroblasts undergo growth, change of shape, multinucleation, and establishment of intercellular junctions. One feature of rodent decidual cells is the accumulation of intermediate filaments. In spite of the fact that fibroblasts normally have vimentin intermediate filaments, they acquire a large amount of desmin intermediate filaments while they undergo decidualization. The light and electron microscope immunocytochemical results of the present work show that during the initial stages of decidual transformation the desmin intermediate filaments accumulate around the nuclei, often forming caps around the nuclear envelope. As the decidual cells grow, the filaments form bundles and nets that radiate from the nuclei toward the cell surface. During the final stages of differentiation, on day 8 of pregnancy, staining of differentiated decidual cells decreases and most filaments accumulate under the cell surface. A role for intermediate filaments is suggested for decidualization of mouse endometrial cells.     

Contribution of the Cytoskeleton to the Compressive Properties and Recovery Behavior of Single Cells

The cytoskeleton is known to play an important role in the biomechanical nature and structure of cells, but its particular function in compressive characteristics has not yet been fully examined. This study focused on the contribution of the main three cytoskeletal elements to the bulk compressive stiffness (as measured by the compressive modulus), volumetric or apparent compressibility changes (as further indicated by apparent Poisson's ratio), and recovery behavior of individual chondrocytes. Before mechanical testing, cytochalasin D, acrylamide, or colchicine was used to disrupt actin microfilaments, intermediate filaments, or microtubules, respectively. Cells were subjected to a range of compressive strains and allowed to recover to equilibrium. Analysis of the video recording for each mechanical event yielded relevant compressive properties and recovery characteristics related to the specific cytoskeletal disrupting agent and as a function of applied axial strain. Inhibition of actin microfilaments had the greatest effect on bulk compressive stiffness (∼50% decrease compared to control). Meanwhile, intermediate filaments and microtubules were each found to play an integral role in either the diminution (compressibility) or retention (incompressibility) of original cell volume during compression. In addition, microtubule disruption had the largest effect on the “critical strain threshold” in cellular mechanical behavior (33% decrease compared to control), as well as the characteristic time for recovery (∼100% increase compared to control). Elucidating the role of the cytoskeleton in the compressive biomechanical behavior of single cells is an important step toward understanding the basis of mechanotransduction and the etiology of cellular disease processes.     

Different regions of bovine deep digital flexor tendon exhibit distinct elastic,but not viscous,mechanical properties under both compression and shear loading

Tendons in different locations function in unique, and at times complex, invivo loading environments. Specifically, some tendons are subjected to compression, shear and/or torsion in addition to tensile loading, which play an important role in regulating tendon properties. To date, there have been few studies evaluating tendon mechanics when loaded in compression and shear, which are particularly relevant for understanding tendon regions that experience such non-tensile loading during normal physiologic function. The objective of this study was to evaluate mechanical responses of different regions of bovine deep digital flexor tendons (DDFT) under compressive and shear loading, and correlate structural characteristics to functional mechanical properties. Distal and proximal regions of DDFT were evaluated in a custom-made loading system via three-step incremental stress-relaxation tests. A two-relaxation-time solid linear model was used to describe the viscoelastic response. Results showed large differences in the elastic behavior between regions: distal region stresses were 4–5 times larger than proximal region stresses during compression and 2–3 times larger during shear. Surprisingly, the viscous (i.e., relaxation) behavior was not different between regions for either compression or shear. Histological analysis showed that collagen and proteoglycan in the distal region distributed differently from the proximal region. Results demonstrate mechanical differences between two regions of DDFT under compression and shear loading, which are attributed to variations of composition and microstructural organization. These findings deepen our understanding of structure–function relationships of tendon, particularly for tissues adapted to supporting combinations of tension, compression, and shear in physiological loading environments.     

Immunohistochemical, autoradiographic and electron microscopic studies on the transformation of fibroblasts into chondrocytes in the mouse subfascia induced by bone morphogenetic protein.

Functional morphology on the transformation of fibroblasts into chondrocytes induced by bone morphogenetic protein (BMP) was studied by light and electron microscopy using 35S autoradiography and immunohistochemistry for S-100 protein and type-II collagen. A pellet containing BMP obtained from a murine osteosarcoma was transplanted into the mouse subfascia. By 3 days after implantation, many typical fibroblasts, which were free of the silver grains for 35S and devoid of both S-100 protein and type-II collagen, entered the pellet region. By 5 days, the fibroblasts in the pellet region became polygonal in shape, and cytoplasmic vesicles and vacuoles appeared, both containing a homogeneous substance of low electron density. At 5 days, autoradiography revealed many silver grains for 35S over the Golgi apparatus and vesicles and vacuoles of the cells in the pellet region as well as over the surrounding extracellular matrix. Moreover, the cells at 5 days displayed immunoreactivity to both proteins. The extracellular matrix around the cell began to show clear metachromasia and increased in amount with time. At 9 days all the cells in the pellet region were round or oval in shape and surrounded by an abundant cartilaginous matrix. The rough endoplasmic reticulum and Golgi apparatus were extremely well developed, and a large number of vacuoles and vesicles were seen in the cytoplasm. These cells showed intense immunoreactivity to both proteins, and strong accumulation of sulfur was visualized in the extracellular matrix by autoradiography. These results suggest that the fibroblasts in the pellet region change into chondroblasts by 5 days, and become typical chondrocytes by 9 days.     

The regional contribution of glycosaminoglycans to temporomandibular joint disc compressive properties

Understanding structure-function relationships in the temporomandibular joint (TMJ) disc is a critical first step toward creating functional tissue replacements for the large population of patients suffering from TMJ disc disorders. While many of these relationships have been identified for the collagenous fraction of the disc, this same understanding is lacking for the next most abundant extracellular matrix component, sulfated glycosaminoglycans (GAGs). Though GAGs are known to play a major role in maintaining compressive integrity in GAG-rich tissues such as articular cartilage, their role in fibrocartilaginous tissues in which GAGs are much less abundant is not clearly defined. Therefore, this study investigates the contribution of GAGs to the regional viscoelastic compressive properties of the temporomandibular joint (TMJ) disc. Chondroitinase ABC (C-ABC) was used to deplete GAGs in five different disc regions, and the time course for >95% GAG removal was defined. The compressive properties of GAG depleted regional specimens were then compared to non-treated controls using an unconfined compression stress-relaxation test. Additionally, treated and non-treated specimens were assayed biochemically and histologically to confirm GAG removal. Compared to untreated controls, the only regions affected by GAG removal in terms of biomechanical properties were in the intermediate zone, the most GAG-rich portion of the disc. Without GAGs, all intermediate zone regions showed decreased tissue viscosity, and the intermediate zone lateral region also showed a 12.5% decrease in modulus of relaxation. However, in the anterior and posterior band regions, no change in compressive properties was observed following GAG depletion, though these regions showed the highest compressive properties overall. Although GAGs are not the major extracellular matrix molecule of the TMJ disc, they are responsible for some of the viscoelastic compressive properties of the tissue. Furthermore, the mechanical role of sulfated GAGs in the disc varies regionally in the tissue, and GAG abundance does not always correlate with higher compressive properties. Overall, this study found that sulfated GAGs are important to TMJ disc mechanics in the intermediate zone, an important finding for establishing design characteristics for future tissue engineering efforts.     

Detection of glial fibrillary acidic protein (GFAP) and vimentin (Vim) by immunoelectron microscopy of the glial cells in the central nervous system of the snail Megalobulimus abbreviatus

When examined under an electron microscope, the central nervous system of Megalobulimus abbreviatus showed two types of glial cells: firstly, protoplasmic glial cells which displayed a nucleus with peripheral heterochromatin, scanty or no intermediate filaments, a developed Golgi complex, rough and smooth endoplasmic reticula, mitochondria and polymorphic lysosomes that indicate phagocytic activity of debris from the extracellular space; and, secondly, fibrous glial cells which showed numerous glial fibrillary acidic protein (GFAP) and vimentin immunoreactive intermediate filament bundles, a discrete Golgi complex, mitochondria, endoplasmic reticulum, lipid droplets and lysosomes. The contacts between the glial cells consisted of desmosomes and puncta adherentia, while those between the glial cells and the basal lamina consisted of hemidesmosomes. Both glial cell types were located in the cortex and medullary regions, however, the protoplasmic glial cells prevailed in the cortical region, while the fibrous glial cells prevailed in the medullar region. As the nervous tissue is avascular, the passage of nutrients and waste products may be facilitated by the glial labyrinthic system which is located in the cortical region. Glial processes adjacent to large and giant neurones formed a trophospongium, which seemed to be involved in a metabolic exchange between these cells. Thus, this evidence suggests that glial cells of M. abbreviatus are involved in structural support, isolation of different ganglionic areas, the formation of a microcirculatory system and an intimate metabolic relationship with neurones.     

Vimentin Enhances Cell Elastic Behavior and Protects against Compressive Stress

Vimentin intermediate filament expression is a hallmark of epithelial-to-mesenchymal transitions, and vimentin is involved in the maintenance of cell mechanical properties, cell motility, adhesion, and other signaling pathways. A common feature of vimentin-expressing cells is their routine exposure to mechanical stress. Intermediate filaments are unique among cytoskeletal polymers in resisting large deformations in vitro, yet vimentin’s mechanical role in the cell is not clearly understood. We use atomic force microscopy to compare the viscoelastic properties of normal and vimentin-null (vim^−/−) mouse embryo fibroblasts (mEFs) on substrates of different stiffnesses, spread to different areas, and subjected to different compression patterns. In minimally perturbed mEF, vimentin contributes little to the elastic modulus at any indentation depth in cells spread to average areas. On a hard substrate however, the elastic moduli of maximally spread mEFs are greater than those of vim^−/−mEF. Comparison of the plastic deformation resulting from controlled compression of the cell cortex shows that vimentin’s enhancement of elastic behavior increases with substrate stiffness. The elastic moduli of normal mEFs are more stable over time than those of vim^−/−mEFs when cells are subject to ongoing oscillatory compression, particularly on a soft substrate. In contrast, increasing compressive strain over time shows a greater role for vimentin on a hard substrate. Under both conditions, vim^−/−mEFs exhibit more variable responses, indicating a loss of regulation. Finally, normal mEFs are more contractile in three-dimensional collagen gels when seeded at low density, when cell-matrix contacts dominate, whereas contractility of vim^−/−mEF is greater at higher densities when cell-cell contacts are abundant. Addition of fibronectin to gel constructs equalizes the contractility of the two cell types. These results show that the Young’s moduli of normal and vim^−/−mEFs are substrate stiffness dependent even when the spread area is similar, and that vimentin protects against compressive stress and preserves mechanical integrity by enhancing cell elastic behavior.