Musculo-tendinous junction of the flexor carpi ulnaris muscle. An anatomical study

The aim of the study was to evaluate the occurrence of the anatomical variations of the musculotendinous junction of the flexor carpi ulnaris (FCU) muscle and the variations of its insertion onto the pisiform. One hundred cadaver specimens preserved according to Thiel’s method were assessed. Following careful dissection, the distance between the musculotendinous junction and the pisiform and the width of the muscle belly were determined. Three typical anatomical variations were found 1) a large muscle belly running distally almost to the insertion onto the pisiform 2) the muscle belly ending more proximally, with some large fibres running parallel to the tendon and almost reaching the pisiform 3) the musculotendinous junction ending more proximally, with only single fibres continuing distally. The length of the tendon was greater than 10 mm. A number of variations of the distal region of FCU were observed. The presence of muscle fibres almost reaching the insertion point onto the pisiform have to be considered when interpreting MRI or ultrasound findings of this region.     

Three-dimensional reconstructions of the Achilles tendon insertion in man

The distribution of type II collagen in sagittal sections of the Achilles tendon has been used to reconstruct the three‐dimensional (3D) shape and position of three fibrocartilages (sesamoid, periosteal and enthesis) associated with its insertion. The results showed that there is a close correspondence between the shape and position of the sesamoid and periosteal fibrocartilages – probably because of their functional interdependence. The former protects the tendon from compression during dorsiflexion of the foot, and the latter protects the superior tuberosity of the calcaneus. When the zone of calcified enthesis fibrocartilage and the subchondral bone are mapped in 3D, the reconstructions show that there is a complex pattern of interlocking between pieces of calcified fibrocartilage and bone at the insertion site. We suggest that this is of fundamental importance in anchoring the tendon to the bone, because the manner in which a tendon insertion develops makes it unlikely that many collagen fibres pass across the tissue boundary from tendon to bone. When force is transmitted to the bone from a loaded tendon, it is directed towards the plantar fascia by a series of highly orientated trabeculae that are clearly visible in 3D in thick resin sections.     

Fibrocartilage at the entheses of the suprascapular (superior transverse scapular) ligament of man—a ligament spanning two regions of a single bone

The suprascapular ligament converts the suprascapular notch into a foramen separating the vessels and nerve of the same name. It connects 2 regions of the same bone and does not cross any joint, and no mechanical function has yet been attributed to it. Nevertheless, variations in its thickness and length, and its tendency to ossify, suggest that the ligament responds to changes in mechanical load. This should be reflected in the composition of the extracellular matrix. The primary purpose of the present study is to demonstrate that the suprascapular ligament has fibrocartilaginous entheses (i.e. insertion sites), even though there is no obvious change in insertional angle that directly results from joint movement. Such a change is more typical of tendons or ligaments that cross highly mobile joints. The complete ligament (including both entheses) was removed from 7 cadavers shortly after death and fixed in 90% methanol. Cryosections were immunolabelled with a panel of monoclonal antibodies against collagens (types I, II, III, VI), glycosaminoglycans (chondroitin 4 sulphate, chondroitin 6 sulphate, dermatan sulphate and keratan sulphates), proteoglycans (aggrecan and versican) and link protein. Both entheses were strongly fibrocartilaginous, and a moderately fibrocartilaginous matrix was also detected throughout the remainder of the ligament. The extracellular matrix of both entheses labelled strongly for type II collagen, aggrecan and link protein. The fibrocartilaginous character of the entheses suggests that the insertion sites of the ligament are subject to both compressive and tensile loading and are regions of stress concentration. This in turn probably reflects the complex shape of the scapula and the presence of a conspicuous indentation (the suprascapular notch) near the ligament. The loading patterns may reflect either the attachment of muscles and/or the forces transmitted to the suprascapular ligament from the neighbouring coracoclavicular ligament.     

Expression of extracellular matrix molecules typical of articular cartilage in the human scapholunate interosseous ligament

The scapholunate interosseous ligament (SLIL) connects the scaphoid and lunate bones and plays a crucial role in carpal kinematics. Its rupture leads to carpal instability and impairment of radiocarpal joint function. As the ligament is one of the first structures affected in rheumatoid arthritis, we conducted an immunohistochemical study of cadaveric tissue to determine whether it contains known autoantigens for rheumatoid arthritis. We immunolabelled the ligament from one hand in 12 cadavers with monoclonal antibodies directed against a wide range of extracellular matrix (ECM) molecules associated with both fibrous and cartilaginous tissues. The labelling profile has also enabled us to comment on how the molecular composition of the ligament relates to its mechanical function. All regions of the ligament labelled for types I, III and VI collagens, chondroitin 4 and 6 sulphates, keratan sulphate, dermatan sulphate, versican, tenascin and cartilage oligomeric matrix protein (COMP). However, both entheses labelled strongly for type II collagen, aggrecan and link protein and were distinctly fibrocartilaginous. In some regions, the ligament attached to bone via a region of hyaline cartilage that was continuous with articular cartilage. Labelling for cartilage molecules in the midsubstance was most evident dorsally. We conclude that the SLIL has an ECM which is typical of other highly fibrocartilaginous ligaments that experience both tensile load and shear. The presence of aggrecan, link protein, COMP and type II collagen could explain why the ligament may be a target for autoantigenic destruction in some forms of rheumatoid arthritis.     

Distribution of the extracellular matrix components in human glomerular lesions

Most glomerular pathologies are associated with alterations of the matrix compartment. Using reagents directed against the α/α2 and α3 chains of type IV collagen [α1/α2(IV), α3(IV)], laminin, heparan sulphate proteoglycan (HPG), fibronectin, collagen I, and collagen III, we investigated the modifications of the glomerular matrix components in several human glomerular lesions compared with normal kidney. In type I membranous glomerulo-nephritis (MGN) (nine cases), we did not observe alterations in the matrix component distribution. In MGN types II and III (five cases), the spikes and chainettes were made of the α3(IV) chain, laminin, and HPG, while the α1/α2(IV) chains were localized along the subendothelial side of the glomerular basement membrane (GBM). In focal and segmental glomerulosclerosis (six cases), fibronectin, α1/α2(IV) chains, laminin, and small amounts of interstitial collagens were detected within the collapsed capillary loops; the newly formed matrix material between the podocytes and the GBM contained the α1/α2(IV) chains, laminin, and HPG but not the α3(IV) chain. In crescentic glomerulo-nephritis (six cases), fibronectin was the most abundant and, in purely cellular crescents, the unique component. A basement membrane-like network containing laminin, HPG, α1/α2(IV) chains, and interstitial collagens developed in a second step between the crescent cells. Interstitial collagens were present in the crescent framework, even when the integrity of Bowman's capsule was preserved. In membranoproliferative glomerulonephritis (five cases), we observed strong accumulation of fibronectin in the thickened mesangial spaces together with accumulation of laminin, α1/α2(IV) chains, and HPG; type I collagen was also present in the central part of the mesangial areas. This study shows that each glomerular lesion is characterized by particular alterations of the matrix components.     

An ultrastructural and immunohistochemical study of microcystic meningioma with emphasis on matrix proteins and connexin 26 type gap junctions

Although the histopathological subtypes of meningioma do not themselves appear to have prognostic significance, they are collectively important for defining the overall histopathological entity of microcystic meningioma (MCM) and allowing a distinction from other intracranial tumors, such as capillary hemangioblastoma, glioma, and metastatic renal cell carcinoma showing similar histology. Four cases of MCM were analyzed by conventional histology, immunohistochemistry, and electron microscopy. The present series of MCM was characterized by spindle- or cobweb-shaped tumor cells, characteristically associated small blood vessels, and a peculiar microcystic pattern. Among the microcystic meningeal tumor tissue, small areas of conventional subtypes were identified. Immunohistochemically, tumor cells showed the mesenchymal features of vimentin positivity and a rich distribution of matrix proteins around tumor cells. They lacked epithelial marker positivity but were faintly EMA positive. Ultrastructurally, primitive cellular junctions, desmosomes, and gap junctions were frequently seen between tumor cells. The gap junctions correlated with connexin 26 immunoreactivity. Although lacking an obvious epithelial nature, these features could be interpreted as showing an abortive differentiation mimicking meningothelial (arachnoidal) cells, which, physiologically, regulate cerebrospinal fluid between blood vessels and brain parenchyma.     

Immunohistochemical localization of interstitial collagens in bone tissue from patients with various forms of osteogenesis imperfecta

Immunohistochemical studies of bone from individuals with osteogenesis imperfecta (OI) type II, OI type III, or OI type IV demonstrate a similar pattern, but varying extent, of the abnormal presence of interstitial collagens in bone matrix. OI type II bone had nests of cartilage with type II collagen, and significant type III collagen in the bone matrix. In OI types III and IV, type II collagen was present only in epiphyseal cartilage but bone still contained type III collagen. These findings resembled those in developing fetal bone indicating the “immature” nature of OI bone.     

An immunohistochemical study of the extracellular matrix of the tarsal plate in the upper eyelid in human beings

The superior tarsus is a plate of tissue that stiffens the upper eyelid, gives it support and determines its form. The purpose of the present study was to relate the composition of its extracellular matrix to its function and to report regional differences that may influence the activity of its Meibomian glands. Fourteen methanol-fixed specimens were cryosectioned for immunohistochemistry and labelled with a panel of monoclonal antibodies against a wide range of collagens, glycosaminoglycans and proteoglycans. Labelling was detected with avidin-biotin-peroxidase. A further six specimens were formalin-fixed for routine histology. The tarsal plate immunolabelled strongly for types I, III and VI collagen and for aggrecan, versican, tenascin, cartilage oligomeric matrix protein (COMP) together with a variety of glycosaminoglycans (notably chondroitin 6 sulphate). A region of strong labelling for aggrecan, dermatan sulphate and chondroitin 6 sulphate immediately surrounded the Meibomian glands. The site of labelling corresponded to a layer of acellular and amorphous matrix seen histologically that we have termed the 'territorial matrix'. The results suggested that the tarsal plate is a specialized connective tissue that is neither purely fibrous nor cartilaginous, yet has an aggrecan content that probably contributes to its stiffness. Its unique character highlights the challenge in choosing an ideal mechanical substitute. As patients with rheumatoid arthritis often have problems relating to tear film deficiency, the ability of aggrecan or COMP to act as autoantigens may be significant. An immune reaction directed against these molecules could alter tarsal gland function by interfering with the interaction between the glands and their territorial matrix.     

An α1 II Gly913 to Cys substitution prevents the matrix incorporation of type II collagen which is replaced with type I and III collagens in cartilage from a patient with hypochondrogenesis

A heterozygous mutation in the COL2A1 gene was identified in a patient with hypochondrogenesis. The mutation was a single nucleotide transition of G3285T that resulted in an amino acid substitution of Cys for Gly⁹¹³ in the α1(II) chain of type II collagen. This amino acid change disrupted the obligatory Gly-X-Y triplet motif required for the normal formation of a stable collagen triple helix and prevented the deposition of type II collagen into the proposita's cartilage, which contained predominantly type I and III collagens and minor amounts of type XI collagen. Biosynthetic analysis of collagens produced and secreted by the patient's chondrocytes cultured in alginate beads was consistent with the in vivo matrix composition, demonstrating that the main products were type I and III collagens, along with type XI collagen. The synthesis of the cartilage-specific type XI collagen at similar levels to controls indicated that the isolated cartilage cells had re-differentiated to the chondrocyte phenotype. The chondrocytes also produced small amounts of type II collagen, but this was post-translationally overmodified and not secreted. These data further delineate the biochemical and phenotypic consequences of mutations in the COL2A1 gene and suggest that cartilage formation and bone development can take place in the absence of type II collagen. © 1996 Wiley-Liss, Inc.     

An immunohistochemical study of the extracellular matrix in oral squamous cell carcinoma and its association with invasive and metastatic potential

The expression of extracellular matrices (ECMs) laminin (LN), type IV collagen (IV C), heparansulphate proteoglycan (HS-PG), fibronectin (FN), tenascin (TN), decorin and vitronectin (VN) was examined immunohistochemically in 112 primary tumours and 29 metastatic cervical lymph nodes in oral squamous cell carcinoma (OSCC). In highly invasive primary tumours, the expression of LN, IV C and HS-PG in the basement membrane along the tumour-stroma borderline and the expression of decorin and VN in the tumour stroma at the invasive site were all significantly decreased. The expression of FN and TN in the tumour stroma at the same site was markedly increased. In peritumour stroma in metastatic lymph nodes, LN, IV C, HS-PG, decorin and VN were weakly expressed, while FN and TN were strongly expressed. Thus, the staining pattern of the ECMs in the metastatic lymph nodes was similar to that in highly invasive primary tumours. Furthermore, in primary tumours of metastatic cases, the expression of LN, IV C, HS-PG, decorin and VN obviously decreased, while the expression of FN and TN increased when compared with those of the non-metastatic cases. The investigation of ECMs in OSCC was valuable in predicting tumour behaviour.     

An immunohistochemical study of enthesis development in the medial collateral ligament of the rat knee joint

The changing distributions of collagens and glycosaminoglycans have been studied at the attachments of the medial collateral ligament during postnatal development. The ligament is of particular interest because it has a fibrocartilaginous attachment to the femoral epiphysis, but a fibrous one to the tibial metaphysis. Ligaments were examined in rats killed at birth and at 2, 4, 6, 8, 10, 20, 30, 45, 60, 90 and 120 days after birth. Cryosections were immunolabelled with monoclonal and polyclonal antibodies against types I and II collagen, chondroitin 4 and 6 sulfate, dermatan and keratan sulfate. Although the ligament is attached at both ends to bones that develop from cartilage, there was a striking difference in collagen labelling. Type II collagen was only found in spicules of calcified cartilage in bone beneath the tibial enthesis after ossification had commenced, but there was a continuous band of labelling at all stages of development at the femoral enthesis. Initially, the cartilage at the femoral attachment lacked type I collagen, but by 45 days labelling was continuous from ligament to bone. Continuity of labelling was seen much earlier at the tibial enthesis, as soon as bone had formed. There were also marked changes in glycosaminoglycan distribution. Keratan sulfate was present at both entheses up to 45 days, but only at the femoral enthesis thereafter. Both attachments labelled throughout life for dermatan sulfate, but chondroitin 4 and 6 sulfate were only found at the femoral end. The results suggest that enthesial cartilage at the femoral attachment was initially derived from the cartilaginous bone rudiment but was quickly eroded on its deep surface by endochondral ossification as bone formed at the attachment site. It was replaced by fibrocartilage developing in the ligament. This mechanism allows enthesis cartilage/fibrocartilage to contribute to the growth of a bone at a secondary centre of ossification in addition to dissipating stress at the ligament-bone junction.     

Osteoblastic differentiation of periosteum‐derived cells is promoted by the physical contact with the bone matrix in vivo

The periosteum contains osteoprogenitors that differentiate to osteoblasts in bone growth or repair. Our previous studies suggested the hypothesis that the physical contact of the periosteum with the bone matrix is requisite for the differentiation of osteoblasts. To test the hypothesis, the present study was designed to investigate how the contact between the periosteum and the bone matrix influences the osteoblastic differentiation of periosteal cells with establishing a new experimental model in vivo. Differentiation of osteoblasts was assessed by gene expression of type I collagen, osteocalcin and bone sialoprotein using in situ hybridization. A barrier was designed to prevent periosteal cells from contacting the bone matrix using the membrane filter. The membrane filter was inserted surgically between the surface of rat parietal bone and the periosteum after being punched out with pin holes. Periosteal cells were allowed to contact with the bone surface only through the pin holes. The pin hole was filled with cells derived from the periosteum 1 week after inserting the filter. Differentiation of osteoblasts in week 2 and noticeable bone formation in week 3 were identified on the bone surface only under the pin hole but not under the filter. The present study demonstrated that the physical contact with the bone matrix promotes osteoblastic differentiation of periosteum‐derived cells in vivo. Anat Rec 264:72–81, 2001. © 2001 Wiley‐Liss, Inc.     

Alteration of the basement membrane in human thyroid diseases: An immunohistochemical study of type IV collagen,laminin and heparan sulphate proteoglycan

Basement membrane (BM) alteration in thyroid diseases was examined by immunohistochemistry using antibodies for the three major BM proteins: type IV collagen, laminin and heparan sulphate proteoglycan. Linear epithelial BMs surrounding follicles accompanied by vascular BMs forming loops, similar to those seen in the normal thyroid, were observed in Graves' disease and adenomatous goitre. Hashimoto's thyroiditis showed scant epithelial BMs as a result of follicle destruction. In follicular adenomas, development of epithelial BMs seemed to be related to follicle formation; well-developed epithelial BMs were frequently seen in normo- or largefollicular type, whereas trabecular or solid types revealed scant or poorly developed epithelial BMs. Lumpy accumulation of BM proteins was detected in hyalinizing trabecular adenomas. Papillary carcinomas revealed two different types of papillae; one type contained both epithelial and vascular BMs, and the other had only vascular BMs. Epithelial BMs in invasive areas of papillary carcinoma were distributed in an irregular, interrupted manner, and were completely absent in many foci. Anaplastic carcinomas showed scant or a total loss of epithelial BMs. These results suggest that alterations of BM in thyroid diseases clearly reflect their architectural variations, presumably in connection with their function and/or biological behaviour.     

Ioss of basement membranes in the invading front of O-1N, hamster squamous cell carcinoma with high potential of lymph node metastasis: An immunohistochemical study for laminin and type IV collagen

The change in immunohistochemical localization of the two basement membrane molecules, laminin and type IV collagen, was studied in relation to tumor growth and lymphatic invasion in the transplanted hamster squamous cell carcinoma, O-1N, that has a high potential of lymph node metastasis. At 1 week after transplantation, the tumors consisted of large round-shaped nests of approximately 200 mm in diameter, 80% of which were encircled by continuous laminin and type IV collagen positive lines representing the basement membrane. At 5 weeks, however, the tumor cell nests became half in diameter with invasion in small islands or cords at the periphery and only 40% of them had continuous basement membrane. At 10 weeks, the basement membranes were disrupted in varying degrees in approximately 90% of the tumor cell nests. The disruption was most conspicuous on the outer and invading side of the nests. Lymphatic invasion and lymph node metastasis were observed in animals after 5 weeks of transplantation and the immunoreactivity was absent around tumor cell clusters growing in lymphatic spaces. The findings indicate that the disappearance of basement membrane and reduction in size of tumor cell nests are essential signs for local invasion of tumor cells leading to lymphatic invasion and metastasis to regional lymph nodes.     

Large cell calcifying Sertoli cell tumor of the testis: comparative immunohistochemical study with Leydig cell tumor

Large cell calcifying Sertoli cell tumor is a rare type of testicular tumor. Reported herein is a Japanese patient with this tumor not associated with Carney's complex. An 11-year-old boy was admitted to hospital because of left testicular enlargement, and radical orchiectomy was performed. Macroscopically, the tumor was well circumscribed and had a maximum diameter of approximately 2 cm. The cut surface showed a yellow-white solid mass. Histologically, the tumor was composed of large neoplastic cells with abundant eosinophilic cytoplasm with a tubular, trabecular, and solid arrangement and loose myxoid stroma with irregularly shaped calcification. Immunohistochemically, the tumor cells were positive for vimentin, S-100 protein, calretinin, inhibin-alpha, melan-A, and CD10, and type IV collagen and laminin were observed in the extracellular matrix around the tumor cells. The distributions of melan-A, CD10, and mitochondria were characteristically patchy; in contrast, they were diffusely distributed in the cytoplasm in a control case of Leydig cell tumor. The differences in immunostaining patterns for melan-A, CD10, and mitochondria as well as positivity for S-100 protein-beta might be useful diagnostic hallmarks of large cell calcifying Sertoli cell tumor for discrimination from Leydig cell tumor.     

A study by immunofluorescence microscopy of the NC1 domain of collagen type IV in glomerular basement membranes of two patients with hereditary nephritis

Summary The NC1 domain of the collagen type IV molecule, the major component of glomerular basement membranes (GBM), consists of dimers and 24 kilodalton (K), 26 K and 28 K monomers in man, and contains the Goodpasture antigen. Serum obtained from patients with Goodpasture's syndrome has been reported not to stain GBM of most male and some female patients with hereditary nephritis (HN) by immunofluorescence (IF) microscopy. In the present study, GBM seen on the renal biopsies of 2 patients (one male and one female) with HN were examined by IF to ascertain whether NC1 monomers were detectable. Three reagents were used: a plasmapheresis fluid (PPF) obtained from a patient who was treated for anti-GBM nephritis (human anti-GBM PPF); a commercial rabbit antibody against human NC1; and a rabbit antibody raised by us against dog NC1, which cross-reacted with human NC1. All 3 reagents detected NC1 determinants in GBM of normal human kidney by IF and reacted with human NC1 by a plate-binding radioimmunoassay (RIA). The human anti-GBM PPF bound to 28 K and 26 K monomer components of NC1 by Western blotting, the rabbit anti-human NC1 antibody bound to 26 K and 24 K monomers, while the rabbit anti-dog NC1 antibody bound only to the 26 K monomer. By IF, the human anti-GBM PPF did not stain GBM of the male patient with HN, but produced segmental staining of GBM (i.e., some GBM stained, while others did not) of the female patient. In contrast, the rabbit anti-NC1 antibodies produced global staining by IF of GBM of both patients. The absence of staining (i.e., global or segmental) seen with the human anti-GBM PPF implied that the 26 K and 28 K monomers of NC1 were either absent from GBM, or were present but altered structurally, leading to a diminution in their immunological reactivity. However, the positive staining observed with the rabbit anti-NC1 antibodies implied that the 26 K monomer was actually present in GBM. Hence, we postulate that the 26 K monomer of NC1 in GBM was structurally altered, and that the 28 K monomer was either absent, or present but altered. These findings suggest that there is an abnormality of more than one monomer of NC1 in GBM of patients with HN.     

A genome-wide association study identifies new genes associated with developmental dysplasia of the hip

Developmental dysplasia of the hip (DDH) is one of the most common congenital malformations and covers a spectrum of hip disorders from mild dysplasia to irreducible dislocation. The pathological mechanisms of DDH are poorly understood, which hampers the development of diagnostic tools and treatments. To gain insight into its disease mechanism, we explored the potential biological processes that underlie DDH by integrating pathway analysis tools and performing a genome-wide association study (GWAS). A total of 406 DDH-associated genes (P < 0.001) were identified by our GWAS using a Chinese Han cohort consisting of 386 DDH cases and 500 healthy controls (Set A). We verified the significant loci (P < 10⁻⁵) in another Chinese Han cohort consisting of 574 DDH patients and 569 healthy controls (Set B). An intronic Single Nucleotide Polymorphism (SNP) (rs61930502) showed significant association in Set A and Set B (P = 2.65 × 10⁻⁷ and 2.0 × 10⁻⁴, respectively). The minor allele, rs61930502-A, which tended to prevent DDH showed a dominant effect. Heat shock 70 kDa protein 8 (HSPA8) showed the most direct interactions with other proteins which were coded by DDH-associated genes in the protein-protein interaction analysis. Interestingly, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested a relation between DDH and the genes involved in type II diabetes mellitus pathway (P = 0.0067). Our genetic and protein interaction evidence could open avenues for future studies of DDH.