Focusing on intramuscular connective tissue: Effect of cooking time and temperature on physical, textual, and structural properties of yak meat.

This study aimed to evaluate the effects of different cooking time (2, 4, and 6 h) and temperature (50, 60, 70, 80, and 90 °C) on physical, textual, and structural properties of longissimus lumborum muscle of yak, and to explore the thermal denaturation process of intramuscular collagen by using a new tool (collagen hybridizing peptide staining, CHP staining). The results showed that tenderness was affected by the interaction of cooking time and temperature and the changes in moisture and collagen composition. In comparison with cooking time, temperature had more obvious effects on cooking loss, moisture content and redness. Scanning electron microscopy showed that as the temperature increased, intramuscular connective tissue gradually degraded, and muscle fibers became more compact. CHP staining showed that the collagen in the perimysium first denatured at 50 °C, and more and more collagen denatured and degraded as the temperature increased.

Animal model with structural similarity to human corneal collagen fibrillar arrangement.

Rabbit and porcine corneas have been used in scientific research due to their structural similarity to the human cornea. Currently, there are no studies that have compared corneal collagen fibrillar diameter, interfibrillar distance and interlamellar distance between human and animal models. Ten pairs of porcine, rabbit, and human corneas were used. These were analysed using light and Transmission Electron microscopy. The collagen fibrillar diameter, interfibrillar distance and interlamellar distance were statistically compared between porcine, rabbit and human corneas. The human, porcine and rabbit; mean collagen fibrillar diameters were: 24.52 ± 2.09 nm; 32.87 ± 0.87 nm; and 33.67 ± 1.97 nm. The mean interfibrillar distances were: 46.10 ± 2.44 nm; 53.33 ± 2.24 nm; and 52.87 ± 2.73 nm, respectively. The collagen fibrillar diameter and interfibrillar distance of porcine and rabbit corneas were significantly different (p < 0.001) to the human corneal values but not form each other. The interlamellar distance of human, porcine and rabbit corneas was: 2190 ± 820 nm; 6460 ± 1180 nm; and 4410 ± 1330 nm, respectively. All the comparisons were statistically different, in porcine versus rabbit at the p < 0.01 level and both porcine and rabbit versus human at the p < 0.001 level. Histologically, all five layers (epithelium, Bowman's layer, stroma, Descemet membrane and endothelium) of the cornea were visible in all the three species. While neither animal model was structurally identical to the human cornea, they are both relatively close to being used as models to study the biomechanical effects of external insults/treatments to be extrapolated to the human cornea.

Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

The lens, suspended in the middle of the eye by tendon-like ciliary zonule fibers and facing three different compartments of the eye, is enclosed in what has been described as the thickest basement membrane in the body. While the protein components of the capsule have been a subject of study for many years, the dynamics of capsule formation, and the region-specific relationship of its basement membrane components to one another as well as to other matrix molecules remains to be explored. Through high resolution confocal and super-resolution imaging of the lens capsule and 3D surface renderings of acquired z-stacks, our studies revealed that each of its basement membrane proteins, laminin, collagen IV, nidogen and perlecan, has unique structure, organization, and distribution specific both to the region of the lens that the capsule is located in and the position of the capsule within the eye. We provide evidence of basal membrane gradients across the depth of the capsule as well as the synthesis of distinct basement membrane lamella within the capsule. These distinctions are most prominent in the equatorial capsule zone where collagen IV and nidogen span the capsule depth, while laminin and perlecan are located in two separate lamellae located at the innermost and outermost capsule domains. We discovered that an extracapsular matrix compartment rich in the connective tissue-like matrix molecules fibronectin, tenascin-C, and fibrillin is integrated with the superficial surface of the lens capsule. Each matrix protein in this extracapsular zone also exhibits region-specific distribution with fibrils of fibrillin, the matrix protein that forms the backbone of the ciliary zonules, inserting within the laminin/perlecan lamella at the surface of the equatorial lens capsule.

Radula formation in two species of Conoidea (Gastropoda).

The radula is the basic feeding structure in gastropod molluscs and exhibits great morphological diversity that reflects the exceptional anatomical and ecological diversity occurring in these animals. This uniquely molluscan structure is formed in the blind end of the radular sac by specialized cells (membranoblasts and odontoblasts). Secretion type, and the number and shape of the odontoblasts that form each tooth characterize the mode of radula formation. These characteristics vary in different groups of gastropods. Elucidation of this diversity is key to identifying the main patterns of radula formation in Gastropoda. Of particular interest would be a phylogenetically closely related group that is characterized by high variability of the radula. One such group is the large monophyletic superfamily Conoidea, the radula of which is highly variable and may consist of the radular membrane with five teeth per row, or the radular membrane with only two or three teeth per row, or even just two harpoon-like teeth per row without a radular membrane. We studied the radulae of two species of Conoidea (Clavus maestratii Kilburn, Fedosov & Kantor, 2014 [Drilliidae] and, Lophiotoma acuta (Perry, 1811) [Turridae]) using light and electron microscopy. Based on these data and previous studies, we identify the general patterns of the radula formation for all Conoidea: the dorsolateral position of two groups of odontoblasts, uniform size, and shape of odontoblasts, folding of the radula in the radular sac regardless of the radula configuration. The morphology of the subradular epithelium is most likely adaptive to the radula type.

Structural Investigation of Epididymal Microvasculature and Its Relation to Telocytes and Immune Cells in Camel.

The vascular and perivascular cells, including telocytes (TCs) and immune cells, play an important role in male fertility. The current study intended to describe in detail the microvascular structures harboring special regulatory devices in addition to the interstitial cellular components of the one-humped camel epididymis. The samples were collected from 10 clinically healthy mature camels (Camelus dromedarius). The distribution and characteristics of TCs, peripheral blood vessels of the epididymis, and immune cells were investigated using the light, immunohistochemistry, immunofluorescence, and transmission electron microscopy analyses. Frequent occlusive or throttle arterioles were demonstrated in the epididymal interstitium and their tunica media consisted of glomus cells. In addition, some vein walls consisted of one or two layers of glomus cells. TCs, fibroblasts, muscle cells, and tunica media of the blood vessels, that present in the loose connective tissue surrounding the intertubular interstitium of camel epididymis, showed a positive reaction with vimentin. The endothelium of blood vessels and veins showed positive immunoreactivity for CD34 and vascular endothelial growth factor (VEGF). Furthermore, VEGF, CD34, and S100 proteins were expressed in dendritic cells (DCs) as well as TCs. The current data suggest the involvement of DCs and TCs in angiogenesis and a possible role for the interstitial components in creating an appropriate milieu for the full maturation of sperms.

Structural and ultrastructural characteristics of the tongue of the Collared Peccary (Pecari tajacu, Linnaeus, 1758).

The tongue is an important organ in species due to its feeding functions, and its structure is influenced by the habitat and diet. The Collared Peccary (Pecari tajacu, Linnaeus, 1758) is a terrestrial mammal that is distributed on the American continents and has an omnivorous diet. This study aimed to describe the morphological characteristics of the tongue, lingual papillae and the connective tissue cores (CTCs) of the Collared Peccary. Eight tongues were collected from the Wild Animals Multiplication Center. The samples (n = 6) were processed for three-dimensional analysis of their dorsal epithelium, and their CTCs by scanning electron microscopy and the other samples (n = 2) were used to observe ultrastructural characteristics by transmission electron microscopy. Filiform papillae were observed in the lingual apex and body with their conical CTC demonstrating ripples in their extent. Two types of fungiform papillae were observed, the first in the apex and body with a dome-shaped CTC and the second forming a dorsolateral line with a radial pattern CTC. The vallate papillae were bilateral in the caudal region with the CTCs, characterised by numerous projections, and foliate papilla were not observed. We found a new type of papilla anteriorly to the vallate papilla with an irregular groove and a CTC formed by triangular-shaped projections with a duct opening at the top. We concluded that the lingual papillae of the Collared Peccary resemble those papillae of other mammals, however, were revealed another form of their CTCs and a new lingual papillae morphological description.

Architecture of connective tissue regenerated by enamel matrix derivative around hydroxyapatite implanted into tooth extraction sockets in the rat maxilla.

We investigated the architecture of periodontal ligament regenerated by an enamel matrix derivative (EMD, Emdogain®) coating on the surface of hydroxyapatite (EMD-HA). Immediately after extraction of the maxillary first molar in rats, HA alone or EMD-HA was implanted into the socket. At 5 days, and 2 and 4 weeks after implantation, the specimens were examined by light and transmission electron microscopy, and immunohistochemistry for periostin and matrix metalloproteinase (MMP)-13. Histological observations revealed a large number of fibroblasts and well-developed blood capillaries in the fibrous connective tissue surrounding EMD-HA at 5 days. Ultrastructural analysis showed a distinct difference in the architecture of the fibrous connective tissue. As compared with the poorly constructed architecture of HA, EMD-HA had an orderly alignment of fibroblasts and bundled collagen fibers, with some fibroblasts in the cytoplasm showing collagen fiber phagocytosis. Periostin immunoreactivity was observed in the fibrous connective tissue around EMD-HA at each time point, but was not seen in HA at 5 days and 2 weeks. MMP-13 immunoreactivity was intensely localized in fibroblasts at 5 days and 2 weeks in EMD-HA. The present results indicate that EMD may greatly contribute to a well-developed architecture accompanied by orderly alignment of fibroblasts and bundled collagen fibers, through accelerated induction of periostin, maintenance of fibrillogenesis, and degradation of collagen fibers by extracellular proteinase and phagocytosis.

Connective Tissue Ultrastructure: A Direct Comparison between Conventional Specimen Preparation and High-Pressure Freezing/Freeze-Substitution.

It is generally agreed within the microscopy community that the quality of ultrastructure within the connective tissue matrix resulting from high-pressure freezing followed by freeze-substitution (HPF/FS) far exceeds that gained following the "conventional" preparation method, which includes aqueous fixation, dehydration, and embedding. Exposure to cryogen at high pressure is the only cryopreservation method capable of vitrifying tissue structure to a depth exceeding 200 µm. Cells within connective tissues prepared by HPF/FS are universally larger, filling the commonly seen void at the juncture between cell and matrix. Without significant shrinkage of cells and the coincident extraction of the cytosolic components, well-resolved organelles are less clustered within an expanded cytosol. Much of the artifact from "conventional" methods occurs as large space filling and also smaller fibril-associated proteoglycans are extracted during fixation. However, the visualization of some matrix features by electron microscopy is actually dependent on the collapse or extraction of these "masking" components. Herein, we argue that an impression of ultrastructure within commonly studied matrices, in particular skin, is best gained following the evaluation of both conventional preparations and tissue prepared by HPF/FS. Anat Rec, 2019. © 2019 American Association for Anatomy.

COL1-related overlap disorder: A novel connective tissue disorder incorporating the osteogenesis imperfecta/Ehlers-Danlos syndrome overlap.

The 2017 classification of Ehlers-Danlos syndromes (EDS) identifies three types associated with causative variants in COL1A1/COL1A2 and distinct from osteogenesis imperfecta (OI). Previously, patients have been described with variable features of both disorders, and causative variants in COL1A1/COL1A2; but this phenotype has not been included in the current classification. Here, we expand and re-define this OI/EDS overlap as a missing EDS type. Twenty-one individuals from 13 families were reported, in whom COL1A1/COL1A2 variants were found after a suspicion of EDS. None of them could be classified as affected by OI or by any of the three recognized EDS variants associated with COL1A1/COL1A2. This phenotype is dominated by EDS-related features. OI-related features were limited to mildly reduced bone mass, occasional fractures and short stature. Eight COL1A1/COL1A2 variants were novel and five recurrent with a predominance of glycine substitutions affecting residues within the procollagen N-proteinase cleavage site of α1(I) and α2(I) procollagens. Selected variants were investigated by biochemical, ultrastructural and immunofluorescence studies. The pattern of observed changes in the dermis and in vitro for selected variants was more typical of EDS rather than OI. Our findings indicate the existence of a wider recognizable spectrum associated with COL1A1/COL1A2.

Evaluation of the Structure of Myodural Bridges in an Equine Model of Ehlers-Danlos Syndromes.

Myodural bridges have been described in various species as connective tissue structures "bridging" small cranio-cervical muscles to the dura. Myodural bridges are thought to stabilize the dural sac during head and neck movements and promote cerebrospinal fluid motion; however, their role in neurological diseases has not yet been established. We report ultrasonographic visualization, necropsy, histopathologic and ultrastructural findings of myodural bridges in horses with hereditary equine regional dermal asthenia (HERDA), an equine model of Ehlers-Danlos syndromes. Five HERDA and 5 control horses were studied. Post-mortem examination and ultrasonographic studies (3 HERDA and 4 controls) demonstrated that the atlanto-occipital and atlanto-axial myodural bridges are dynamic structures "moving" the dura. En block resection of the myodural bridges (4 HERDA and 5 controls) was accomplished and histopathology showed myofiber degeneration in 3 HERDA horses and 1 control. Ultrastructural examination revealed loosely packed collagen fibrils with abnormal orientation in all HERDA horses compared to mild abnormalities in 2 controls. Our study provides necropsy and ultrasonographic evidence of the dynamic aspect of the myodural bridges as dural sac stabilizers. Myodural bridges may be pathologically altered in connective tissue disease as evidenced by the ultrastructural morphology in the HERDA myodural bridge.

The Role of Schlemm's Canal Endothelium Cellular Connectivity in Giant Vacuole Formation: A 3D Electron Microscopy Study.

Purpose: We investigated whether cellular connectivity between Schlemm's canal (SC) inner wall (IW) endothelium, and juxtacanalicular connective tissue (JCT), and between IW endothelial cells, plays a role in giant vacuole (GV) and pore formation by comparing perfusion- and immersion-fixed eyes. Methods: Normal human donor eyes (n = 4) were either immersion-fixed (0 mm Hg) or perfusion-fixed (15 mm Hg). Trabecular meshwork near SC was imaged using serial block-face scanning electron microscopy. A total of 12 IW cells from each group were 3D-reconstructed from ∼7040 electron micrographs and compared. In each cell, connections between IW cells and JCT cells/matrix were quantified; IW/IW connectivity was measured by cell border overlap length. GV volume, density, shape, and intracellular and paracellular pores were analyzed. Results: The mean number of IW/JCT cell-cell connections per cell significantly decreased (P < 0.01) while the summed GV volume per cell significantly increased (P < 0.01) in perfusion-fixed eyes compared to immersion-fixed eyes. Intracellular pores were observed in 14.6% of GVs in perfusion-fixed eyes and not observed in immersion-fixed eyes. The mean IW/IW overlap length per cell decreased (P < 0.01), and paracellular pores were found only in regions where IW/IW connectivity was minimal (overlap length = 0 µm) in perfusion-fixed eyes and not observed in immersion-fixed eyes. Conclusions: Our data suggest that changes in IW/JCT connectivity may be an important factor in the formation of larger GVs, and decreased IW/IW connectivity may promote paracellular pore formation. Targeting the IW/JCT and IW/IW connectivity may therefore be a potential strategy to regulate outflow resistance and IOP. .

Comparative morphology of the lingual papillae and their connective tissue cores in the tongue of the Abyssinian black-and-white colobus (Colobus guereza).

We observed the morphology of the lingual papillae (filiform, fungiform, foliate, and vallate) and their underlying connective tissue cores (CTCs) in Abyssinian black-and-white colobus monkeys using light and scanning electron microscopy. The tongues of both juvenile and senescent individuals were relatively short in the rostro-caudal direction, with a rounded apex. Lingual tori were absent. Numerous filiform papillae were distributed over the entire tongue, except at the lingual root. A pair of foliate papillae was present on both the lateral and caudal margins of the corpus. Three vallate papillae were distributed on the boundary between the caudal part of the body and the root in both juvenile and senescent individuals. Based on scanning electron microscopy observations, the morphologies of the filiform papillae differed between juvenile and senescent individuals. The epithelial surface of juvenile filiform papillae had a main process, but the associated processes were weak and the underlying CTCs displayed immature morphology. In contrast, the epithelial surface of senescent filiform papillae was associated with several accessory processes, and their underlying CTCs consisted of several auxiliary cores that nearly encircled the main core, forming a concavity in the papilla. CTCs of the filiform papillae showed variable morphology. Juvenile filiform CTCs exhibited a rather primitive morphology, resembling those of the hamster, mole, and Cape hyrax while, conversely, despite the basically folivorous diet of the Abyssinian black-and-white colobus, senescent filiform CTCs resembled those found in omnivorous primates, including members of the Callitrichinae and Homoidea, and also those in Carnivora (e.g., Canidae and Felidae).

Combined effects of aging and low temperature, long time heating on pork toughness.

The combined effects of aging and low temperature, long time heating (LTLT) on meat toughness were investigated. Pork loins were heated at 53 or 58 °C for up to 20 h, and shear force values, cooking loss, moisture content, collagen solubility, electrophoresis of myofibrillar proteins were determined. Structural changes in perimysium were also observed by light microscopy and scanning electron microscopy (SEM). Results showed that aging and LTLT cooking independently affected meat toughness, and higher temperature or longer time were required to decrease toughness of one-day aged meat to the same level as in 10-day aged meat. Collagen solubilization is suggested as the main reason for the tenderization effect of LTLT. Myofibrillar proteolysis might not occur during LTLT cooking, and will not be contributing to meat tenderness.

Spin locking in liquid entrapped in nanocavities: Application to study connective tissues.

Study of the spin-lattice relaxation in the spin-locking state offers important information about atomic and molecular motions, which cannot be obtained by spin lattice relaxation in strong external magnetic fields. The application of this technique for the investigation of the spin-lattice relaxation in biological samples with fibril structures reveals an anisotropy effect for the relaxation time under spin locking, T1ρ. To explain the anisotropy of the spin-lattice relaxation under spin-locking in connective tissue a model which represents a tissue by a set of nanocavities containing water is used. The developed model allows us to estimate the correlation time for water molecular motion in articular cartilage, τc=30µs and the averaged nanocavity volume, V≃5400nm3. Based on the developed model which represents a connective tissue by a set of nanocavities containing water, a good agreement with the experimental data from an articular cartilage and a tendon was demonstrated. The fitting parameters were obtained for each layer in each region of the articular cartilage. These parameters vary with the known anatomic microstructures of the tissue. Through Gaussian distributions to nanocavity directions, we have calculated the anisotropy of the relaxation time under spin locking T1ρ for a human Achilles tendon specimen and an articular cartilage. The value of the fitting parameters obtained at matching of calculation to experimental results can be used in future investigations for characterizing the fine fibril structure of biological samples.

Reappraising the microscopic anatomy of human testis: identification of telocyte networks in the peritubular and intertubular stromal space.

Telocytes are a recently described stromal cell type widely distributed in various organs including the female and male reproductive systems. This study was aimed to investigate for the first time the existence, distribution and characteristics of telocytes in normal human testis by an integrated morphological approach (immunohistochemistry, immunofluorescence and transmission electron microscopy). We found that telocytes displaying typical long and moniliform prolongations and coexpressing CD34 and PDGFRα formed networks in the outer layer of peritubular tissue and around Leydig cells and vessels in the intertubular stroma. Testicular telocytes were immunophenotypically negative for CD31, c-kit/CD117 as well as α-SMA, thus making them clearly distinguishable from myoid cells/myofibroblasts located in the inner layer of peritubular tissue. Transmission electron microscopy confirmed the presence of cells ultrastructurally identifiable as telocytes (i.e. cells with telopodes alternating podomers and podoms) in the aforementioned locations. Intercellular contacts between neighboring telocytes and telopodes were observed throughout the testicular stromal compartment. Telopodes intimately surrounded and often established close contacts with peritubular myoid cells/myofibroblasts, Leydig cells and vessels. Extracellular vesicles were also frequently detected near telopodes. In summary, we demonstrated that telocytes are a previously neglected stromal component of human testis with potential implications in tissue homeostasis deserving further investigation.

Changes in the Structure and Cell Composition of Human Carinal Lymph Nodes during Aging.

Changes in the structure and cell composition of carinal lymph nodes were studied in humans during aging. Replacement of node parenchyma with fibrous connective tissue progressing with age was demonstrated. The medullary matter significantly prevailed over the cortical substance. The lymph nodes in the cortical substance were small and had no light centers; the concentration of mature CD20+ B cells was high; the paracortical area was fragmented and thinned and contained no CD4+ T helpers. Ki-67+ cells were absent in all structural components of the lymph nodes reflecting exhaustion of lymphopoietic function, which was determined by the replacement of the reticular tissue of the microenvironment with the connective tissue and by the absence of CD4+ T cells regulating cellular and humoral immunity. The disintegration of the reticular stroma in the sinus system that acts as a biological filter impairs the function of lymph purification.

The ultrastructure of spinal cord perivascular spaces: Implications for the circulation of cerebrospinal fluid.

Perivascular spaces play a pivotal role in the exchange between cerebrospinal and interstitial fluids, and in the clearance of waste in the CNS, yet their precise anatomical components are not well described. The aim of this study was to characterise the ultrastructure of perivascular spaces and their role in the transport of fluid, in the spinal cord of healthy rats, using transmission electron microscopy. The distribution of cerebrospinal fluid tracers injected into the subarachnoid space was studied using light, confocal and electron microscopy. Perivascular spaces were found around arterioles and venules, but not capillaries, throughout the spinal cord white and grey matter. They contained fibroblasts and collagen fibres, and were continuous with the extracellular spaces of the surrounding tissue. At 5 min post injection, tracers were seen in the subarachnoid space, the peripheral white matter, the perivascular spaces, basement membranes, extracellular spaces of the surrounding tissue, and surprisingly, in the lumen of blood vessels, suggesting trans-vascular clearance. These findings point out an unrecognised outflow pathway for CNS fluids, with potential implications for volume regulation in health and disease states, but also clinically for the detection of CNS-derived biomarkers in plasma, the immune response and drug pharmacokinetics.

A single stranded fluorescent peptide probe for targeting collagen in connective tissues.

We herein report the construction of a novel single stranded fluorescent collagen mimetic peptide by introducing a bulky FAM dye in the central region rather than the N terminus. Without the need for any prior thermal or ultraviolet treatment, the peptide probe can be conveniently applied to specifically target collagen in connective tissues for fluorescence imaging.

New insights into the cellular makeup and progenitor potential of palatal connective tissues.

The present study investigated the regenerative potential of connective tissues harvested from two palatal areas widely used as donor sites for muco-gingival surgical approaches. Connective tissue grafts (CTGs) were obtained by de-epithelialisation of a free gingival graft (deCTG) and by a split flap approach from a previous donor site (reCTG). Two types of mesenchymal stem cell (MSCs) were isolated and were named de-epithelialised MSCs (deMSCs) and re-entry MSCs (reMSCs). The cells were characterised and cellular functionality was investigated. CTGs were evaluated using immunohistochemical and ultrastructural approaches. No significant differences were observed regarding the frequency of colony-forming unit- fibroblasts, migration potential, and population doubling time between the two cell lines (p > 0.05). Both cell lines showed positivity for CD105, CD73, CD90, and CD44 and negative expression for CD34/45, CD14, CD79a, and HLA-DR. MSCs from both cell lines successfully differentiated into osteogenic, adipogenic, and chondrogenic lineages. Cells expressing antigens characteristic of CD34+ stromal cells (CD34+, αSMA-, CD31-) were traced in both CTGs. Ultrastructural analysis highlighted the presence of putative progenitors, namely fibroblasts,-in the pericapillary regions and in remote regions of the lamina propria- and pericytes-surrounding the capillaries. This study provides supplementary arguments for the use of CTG grafts in clinical practice due to the presence of putative progenitor cell. However, results were inconclusive regarding clinical decision-making to determine optimal harvesting area. Prior harvesting in the donor area did not appear to alter the regenerative capabilities of the connective tissue.

Mechanical adaptability of sea cucumber Cuvierian tubules involves a mutable collagenous tissue.

Despite their soft body and slow motion, sea cucumbers present a low predation rate, reflecting the presence of efficient defence systems. For instance, members of the family Holothuriidae rely on Cuvierian tubules for their defence. These tubules are normally stored in the posterior coelomic cavity of the animal, but when the sea cucumber is threatened by a potential predator, they are expelled through the cloacal aperture, elongate, become sticky and entangle and immobilise the predator in a matter of seconds. The mechanical properties (extensibility, tensile strength, stiffness and toughness) of quiescent (i.e. in the body cavity) and elongated (i.e. after expulsion) Cuvierian tubules were investigated in the species Holothuria forskali using traction tests. Important mechanical differences were measured between the two types of tubules, reflecting adaptability to their operating mode: to ease elongation, quiescent tubules present a low resistance to extension, while elongated tubules present a high toughness to resist tractions generated by the predator. We demonstrate that a mutable collagenous tissue (MCT) is involved in the functioning of these organs: (1) some mechanical properties of Cuvierian tubules are modified by incubation in a cell-disrupting solution; (2) the connective tissue layer encloses juxtaligamental-like cells, a cell type present in all MCTs; and (3) tensilin, a MCT stiffening protein, was localised inside these cells. Cuvierian tubules thus appear to enclose a new type of MCT which shows irreversible stiffening.