Sheena F. McClure

The normal human chondro-osseous junctional region: evidence for contact of uncalcified cartilage with subchondral bone and marrow spaces.

BackgroundThe chondro-osseous junctional region of diarthrodial joints is peculiarly complex and may be considered to consist of the deepest layer of non-calcified cartilage, the tidemark, the layer of calcified cartilage, a thin cement line (between the calcified cartilage and the subchondral bone) and the subchondral bone. A detailed knowledge of the structure, function and pathophysiology of the normal chondro-osseous junction is essential for an understanding of the pathogenesis of osteoarthrosis.MethodsFull thickness samples from human knee joints were processed and embedded in paraffin wax. One hundred serial sections (10 μm thick) were taken from the chondro-osseous junctional region of a block from the medial tibial plateau of a normal joint. They were stained with haematoxylin and eosin and photographed. For a simple physical reconstruction images of each 10th sequential tissue section were printed and the areas of the photomicrographs containing the chondro-osseous junctional region were cut out and then overlaid so as to create a three-dimensional (3D) model of this region. A 3D reconstruction was also made using computer modelling.ResultsHistochemical staining revealed some instances where prolongations of uncalcified cartilage, delineated by the tidemark, dipped into the calcified cartilage and, in places, abutted onto subchondral bone and marrow spaces. Small areas of uncalcified cartilage containing chondrocytes (virtual islands) were seen, in two-dimensional (2D) sections, to be apparently entombed in calcified matrix. The simple physical 3D reconstruction confirmed that these prolongations of uncalcified cartilage were continuous with the cartilage of zone IV and demonstrated that the virtual islands of uncalcified cartilage were cross-sections of these prolongations. The computer-generated 3D reconstructions clearly demonstrated that the uncalcified prolongations ran through the calcified cartilage to touch bone and marrow spaces and confirmed that the apparent entombment of chondrocytes was a 2D artefact.ConclusionThis study demonstrates that the chondro-osseous junctional region is more complex than previously described. The tidemark is a clearly defined boundary delineating uncalcified from calcified cartilage. It is not a straight line across a joint, but a complex three-dimensional structure that follows uncalcified cartilage prolongations dipping down through the calcified cartilage to abut onto subjacent bone or marrow spaces.

The tidemark of the chondro-osseous junction of the normal human knee joint

SummaryThe chondro-osseous junction includes the junction between calcified and non-calcified cartilage matrices often referred to as the tidemark. A detailed knowledge of the structure, function and pathophysiology of the chondro-osseous junction is essential for an understanding both of the normal elongation of bones and of the pathogenesis of osteoarthrosis. In this study the molecular anatomy of the tidemark was studied using histochemical techniques, including lectin histochemistry, on blocks of normal cartilage from human knee joints. The tidemark stained with H&E, picro-sirius red, toluidine blue, safranin O and methyl green, but not with alcian blue in the presence of magnesium chloride at 0.05 M or above. It stained with only four lectins, those from Datura stramonium, Maclura pomifera, Erythrina crystagalli and Helix pomatia, out of the 19 used. Therefore, it is rich in collagen and contains hyaluronan, but appears to lack the glycosaminoglycans of ‘conventional’ proteoglycans and it expresses a very limited and distinctive lectin staining glycoprofile, which is probably attributable to specific glycoproteins. In addition, the tidemark had a distinct microanatomical trilaminate appearance. From all of these results it is clear that this part of the chondro-osseous junctional region is chemically more complex and distinctive than has previously been described.

Lectin Histochemistry of Normal Human Lung

This study aimed to identify and specify the glycotypes of cell populations in normal human lung including types I and II pneumocytes, alveolar macrophages and mast cells, and also in the larger tissue structures of lung, including blood vessels and bronchi/bronchioles, using lectin- and immuno-histochemistry on paraffin-embedded tissue from 11 normal cases. The alveolar macrophages were anti-CD68 positive whereas the cells lining the alveolar walls were positive for cytokeratins. The alveolar macrophages in normal lung tissues showed a broad spectrum of staining for different subsets of N-linked saccharides, N-acetylgalactosamine, N-acetylglucosamine, terminal β-D-galactose and sialyl groups. This study showed that some lectins could be used as specific markers for some cell types i.e. Galanthus nivalis and Narcissus pseudonarcissus lectins for macrophages, Psophocarpus tetragonolobus lectin-II for capillary endothelium, Dolichos biflorus agglutinin for bronchial epithelial cells, Lycopersicon esculentum, Phytolacca americana or Triticum vulgaris (succinylated) for type I pneumocytes and Hippeastrum hybrid or Maclura pomifera lectins for type II pneumocytes. Patchy staining of type I pneumocytes by peanut agglutinin indicated the possibility of two distinct populations of these cells or a pattern of differentiation that is unapparent morphologically.

Lectin histochemistry of normal human gastric mucosa

Information about the saccharides expressed in gastric mucosa is mostly limited to the glycan content of gastric mucins and there are only a few studies of the glycoprofiling of the constituent cells and their components. Knowledge of the glycan expression of normal gastric mucosa is necessary for the interpretation of the significance of changes of expression in disease.A lectin histochemical study of normal human gastric (body) mucosa was performed using 27 lectins chosen to probe for a wide range of oligosaccharide sequences within several categories of glycoprotein glycans.There were marked differences in staining reactions in the various microanatomical structures of the mucosa, particularly between pits and glands with the former more closely resembling the surface epithelium. A notable feature was the degree of difference in the staining between a substantial sub-population of cells within the neck region and the epithelium of both the pits and glands. These neck cells resembled the pit cells with some lectins, glandular cells with some others and neither with some other lectins. Overall, the differences between the pit, gland and neck epithelia were diverse and numerous, and could not be explained by altered activity of a small set of glycosyltransferases. Widespread alterations of glycans must have occurred (affecting terminal and internal parts of their structures) and the very different glycotypes of the pit, neck and gland epithelia are, therefore, suggestive of the existence of three cell lineages within normal gastric epithelium. Published in 2004.

Lectin and other histochemical studies of the articular cartilage and the chondro-osseous junction of the normal human knee joint

There are few studies on normal, adult diarthrodial joints which look in detail at the histochemical properties of the chondro-osseous junctional region. This study of the normal human knee joint was performed using lectin and other histochemical techniques. There were differences in the reactions of mineralised cartilage compared to those of hyaline cartilage with the former demonstrating more collagen and less glycosaminoglycans. Lectin histochemistry revealed more accessible terminal 2-deoxy,2-acetamido-α-d-galactose and more N-acetyllactosamine but less fucosyl and α-2,6-linked-sialyl termini in the mineralised cartilage. The hyaline cartilage chondrocytes stained for N-glycans but those of mineralised cartilage did not. The staining patterns of prolongations and islands of uncalcified cartilage running through the calcified layer to abut bone and marrow spaces were distinct, resembling the patterns of the hyaline cartilage but with some unique features. A possible relationship was revealed between the presence of the Maclura pomifera ligand (Galβ1,3GalNAcα1-) and mineralisation. Subchondral bone had a markedly restricted glycoprofile.

A comparative study of lectin binding to cultured chick sternal chondrocytes and intact chick sternum

Cultured chondrocytes derived from the caudal and cephalic ends of embryonic chick sterna have been compared with each other and with whole sternum, by using a panel of 21 lectins to probe the distribution of oligosaccharides in glycoconjugates of cells and matrix at various times of culture or development. On culture in collagen gels, the cells changed their morphology with time, degrading glycan in the surrounding culture medium and depositing new matrix, the glycan content of which reflected the site of origin of the cells, indicating that the glycan phenotype of both cells and matrix (‘glycotype’) was predetermined and persistent. Sterna of embryonic chicks showed unexpected complexity in their distribution pattern of glycan, containing at least six distinct regions. Major regional temporal differences were evident among saccharides terminating in α-N-acetyl galactosamine and β-galactose, while changes in glycans terminating in fucose, sialic acid and α-mannose were somewhat less marked. Subsets of complex N-glycans changed little.

Glycophenotype of prostatic carcinomas

The factors that affect the progression of prostatic carcinoma are poorly understood, but it is known that carbohydrate antigens on the tumour cell surface play a role in the transforming and metastatic processes. The present report aimed to perform a comparative, lectin-histochemical study of benign and carcinomatous prostates, using a battery of 15 lectins, in combination with monoclonal antibodies against Lewis antigens, and a semi quantitative study, to investigate the changes in glycosylation patterns that occur in prostatic carcinoma. Blocks from 27 necropsy cases of prostatic carcinoma were sectioned and stained with H&E, 15 biotinylated lectins chosen to probe for a wide range of oligosaccharide sequences within several categories of glycoprotein glycans, using a lectin-biotin avidin–peroxidase method, and monoclonal antibodies against Lewisa, sialyl Lewisa and sialyl Lewisx antigens. The glycophenotype of prostatic carcinoma differed from that of the noncancerous prostate in revealing more intense staining with the following lectins (AAA, UEA-1, DBA, WFA, VVA, HPA, BSA-1B4, MPA, ECA, AHA and CTA), while the binding patterns of (GNA and NPA) were almost similar in both prostatic carcinoma and the noncancerous prostate. Lewis antigens are found to be expressed in prostatic carcinomas but not in the noncancerous prostate. The observations of this study suggest that the gylcophenotype of transformed prostatic cells was modified. It showed a moderate increase in, and changing patterns of, fucosylation and galactosylation, increased branching of side chains and sharp rise in 2 deoxy, 2 acetamido galactosylation and masking process by sialylation, especially by α2–3 and α2–6 linkages. O-glycans seems to play an important role in the glycosylation patterns found in prostate carcinoma cells.

Glycophenotype of bone metastases of prostatic carcinomas

The significant morbidity and mortality associated with prostate cancer can universally be attributed to the consequences of metastases. Among prostatic carcinomas, there is a subset of neoplasms which invade bone, while other subsets do not, at least before the patient dies. Those tumours that can invade bone form a prognostically adverse group, but they cannot yet be identified before metastasis to bone has occurred. Therefore, markers capable of predicting tumour progression are required to avoid unnecessary treatment. This study aimed to define the changes in glycan expression occurring during the progression of metastatic prostatic carcinoma and investigate the biological role of fucosylated and sialylated glycans in the progression of prostatic carcinoma and its metastases, especially to bone. Blocks from 29 necropsy cases of prostatic carcinoma, including 62 deposits, were sectioned and stained with H&E, five biotinylated lectins: AAA, UEA-1, DBA, MAA and SNA using a lectin-biotin avidin-peroxidase method, and monoclonal antibodies against prostate specific antigen, Lewisa, sialyl Lewisa and sialyl Lewisx antigens. The results showed differences in the glycophenotype between the primary sites and the metastases. Those cells that metastasised to bone had a distinct glycophenotype, which was demonstrated by the absence or masking of almost all fucosyl residues (i.e. not recognised by AAA and UEA-1). The prostatic cancer cells in sclerotic bone metastases failed to demonstrate Lewis antigens, which were present in all the corresponding primary lesions. On the other hand, non-bone metastases showed a variable expression of the antigens both in the primary lesion and in the metastatic deposits. The findings of this study showed that bone metastasis is associated with two very different glycan phenotypes, the first in the primary tumours and the second in the bone metastases. This study also showed that prostatic metastatic tumours apart from bone metastases have glycophenotype that, in some metastases, are similar to those of the primary sites while in others they are similar to that of the bone metastases.