Electron microscopy of bone.

Mineralized tissues like bone, dentin and mineralized cartilage are difficult to prepare for ultrastructural analysis. In general, the higher the level of mineralization is, the more difficult it is to obtain ultrathin sections of these tissue. Tissues with a low level of mineral, e.g. from young animals, are rather simple to prepare and sectioning is not that much of a problem. In the present chapter we describe step-by-step how to prepare mineralized tissues for ultrastructural examination.

[Spontaneous dental abscesses in familial hypophosphatemic rickets]. / Multipele spontane odontogene abcessen bij familiaire hypofosfatemische rachitis.

Symptoms of familial hypophosphatemic rickets are growth retardation, the formation of O- or X-legs, pain of the joints, spontaneous dental abscesses, and delayed tooth eruption. The dental symptoms are predominantly attributable to the demineralization of dentin. In absence of adequate preventive measurements,familial hypophosphatemic rickets may lead to spontaneous pulpal necrosis. The prophylactic application of occlusal sealants might be effective in preventing abscess formation.

Rapid combined light and electron microscopy on large frozen biological samples.

The use of large unfixed frozen tissue samples (10 x 10 x 5 mm(3)) for combined light microscopy (LM) and electron microscopy (EM) is described. First, cryostat sections are applied for various LM histochemical approaches including in situ hybridization, immunohistochemistry and metabolic mapping (enzyme histochemistry). When EM inspection is needed, the tissue blocks that were used for cryostat sectioning and are stored at -80 degrees C, are then fixed at 4 degrees C with glutaraldehyde/paraformaldehyde and prepared for EM according to standard procedures. Ultrastructurally, most morphological aspects of normal and pathological tissue are retained whereas cryostat sectioning at -25 degrees C does not have serious damaging effects on the ultrastructure. This approach allows simple and rapid combined LM and EM of relatively large tissue specimens with acceptable ultrastructure. Its use is demonstrated with the elucidation of transdifferentiated mouse stromal elements in human pancreatic adenocarcinoma explants grown subcutaneously in nude mice. Combined LM and EM analysis revealed that these elements resemble cartilage showing enchondral mineralization and aberrant muscle fibres with characteristics of skeletal muscle cells.

Involvement of V-ATPases in the digestion of soft connective tissue collagen.

The contribution of vacuolar H+-ATPases (V-ATPases) to collagen degradation was investigated in soft connective tissue explants (periosteum). Immunolocalisation showed faint to intense staining of cells throughout the periosteum. The V-ATPase inhibitors, bafilomycin A1 and folimycin, decreased overall collagen degradation by 40 and 50% after 24 and 48 h, respectively. The participation of V-ATPases in intracellular degradation of collagen was demonstrated by the decrease of the amount of phagocytosed collagen in fibroblasts upon inhibition of pump activity. The inhibition of degradation was not due to a reduction in activity of gelatinase A, an enzyme previously found to mediate collagen degradation, as assessed by zymographic analysis of tissue and conditioned medium. Bafilomycin A1 even induced an increase of gelatinase A and B levels in both fractions. In conclusion, acidification by V-ATPases may represent an important mechanism in extracellular and intracellular collagen degradation in soft connective tissue.

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants.

The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen degradation, as measured by the release of hydroxyproline, decreased significantly on inhibition of the intracellular pool of cysteine proteinases by Ep453. This inhibitor also induced an accumulation of intracellular fibrillar collagen in fibroblasts, indicating a decreased degradation of phagocytosed collagen. The extracellular inhibitor, Ep475, had minor or no effects. Histochemical analysis using a substrate for the cysteine proteinases cathepsins B and L revealed a high level of enzyme activity, which was completely blocked in explants preincubated with a selective intracellular inhibitor of cathepsin B, Ca074-Me. Moreover, the cathepsin B inhibitor strongly affected collagen degradation, decreasing the release of hydroxyproline and increasing the accumulation of phagocytosed collagen. These effects were comparable or slightly stronger than those found with the general intracellular inhibitor (Ep453). Taken together, these data strongly suggest that intracellular cysteine proteinases, in particular cathepsin B, play an important role in the digestion of soft connective tissue collagen.

Ectopic mineralized cartilage formation in human undifferentiated pancreatic adenocarcinoma explants grown in nude mice.

Mineralized as well as nonmineralized cartilage-like structures enclosing cells resembling chondrocytes were found in human-derived undifferentiated but not in poorly differentiated pancreatic adenocarcinoma explants grown in nude mice. The structures reacted with anti-mouse IgG but not with antibodies against human cytokeratin 19, indicating that the newly formed tissue was of mouse origin. High activity of alkaline phosphatase was found in cell layers surrounding the structures and in cells embedded in the matrix. The extracellular matrix was strongly positive after Sirius red staining, reacted with anti-collagen type II antibodies, and the presence of proteoglycans was demonstrated with Alcian blue staining and by metachromasia after Giemsa staining. Electron microscopic inspection revealed the presence of bundles of both thick collagenous fibrils with low levels of fine filamentous material and thin collagenous fibrils with high concentrations of filamentous components. The majority of both types of matrices was found to be partially or completely calcified. The mean area density of the cartilage-like structures in the undifferentiated tumors was 0.31%. The frequent formation of the cartilage-like structures in the rapidly growing undifferentiated explants and its absence in the slowly growing, more differentiated explants suggest that low oxygen tensions in combination with altered levels of growth factors, such as members of the transforming growth factor beta superfamily, create conditions that induce differentiation of fibroblasts to chondrocytes. It is concluded that these human tumors grown in nude mice can be used as an in vivo model to study ectopic formation of mineralized cartilage.

Use of frozen biologic material for combined light and electron microscopy.

A simple and rapid method that enables the use of unfixed frozen material for light and electron microscopic purposes is described. At the light microscopic (LM) level, unfixed cryostat sections were used for enzyme histochemistry. When electron microscopic (EM) inspection was needed, tissue blocks, which were stored at -80 degrees C, were fixed at 4 degrees C and prepared for EM according to standard procedures. Ultrastructural analysis of this material demonstrated that most morphologic aspects of normal (human pancreas and rat liver) and pathologic (human pancreatic adenocarcinoma and rat colon carcinoma metastases in liver) tissue were rather well retained. Cryostat sectioning at -25 degrees C did not appear to have damaging effects on the morphology. The method was applied to correlate enzyme histochemical (LM) data with ultrastructural (EM) aspects of mineralization of stroma in explants of human pancreatic adenocarcinoma grown in nude mice and of nonparenchymal cells around metastases of colon carcinoma in rat liver.

Lymphocyte depletion in thymic nurse cells: a tool to identify in situ lympho-epithelial complexes having thymic nurse cell characteristics.

In situ pre-existing complexes of epithelial cells and thymocytes having thymic nurse cell characteristics were visualized in the murine thymus cortex using dexamethasone as a potent killer of cortisone-sensitive thymocytes. The degradation and subsequent depletion of cortisone-sensitive thymocytes enclosed within cortical epithelial cells appeared to be paralleled by thymocyte degradation and depletion in thymic nurse cells isolated from thymic tissue fragments from dexamethasone-treated animals. This suggests that thymic nurse cells are derived from pre-existing sealed complexes of cortical epithelial cells and thymocytes. Not all thymocytes situated within in situ epithelial or thymic nurse cells complexes appear to be cortisone-sensitive: a minority of 1-2 thymocytes per complex survives the dexamethasone-treatment, thus constituting a minor subset of cortical cortisone-resistant thymocytes predominantly localized within cortical epithelial cells in situ and within thymic nurse cells derived from such structures. Cortisone resistance in thymocytes thus seems to be acquired within the cortical epithelial cell microenvironment. Cortisone-resistant thymocytes in thymic nurse cells express the phenotype of mature precursors of the T helper lineage, indicating that the in situ correlates of thymic nurse cells may play an important role in T cell maturation and selection.

Movement of fibroblasts in the periodontal ligament of the mouse incisor is related to eruption.

Movement of fibroblasts in the periodontal ligament of the lower incisor of the mouse was studied by pulse-labeling with tritiated thymidine and proline. 3H-Thymidine was administered to mark the nuclei of the cells in the proliferative compartment near the basal end of the tooth; 3H-proline gave rise to a narrow band of radioactivity in the dentin, which served as a reference line for measurement of eruption. One or three weeks after injection in each animal, the lower right incisor was prevented from further eruption by being pinned to its alveolar process. The animals were killed 0, 1, or 2 weeks later, and their mandibles processed for LM-radioautography. It was found that in the left incisors, which were not inhibited in their eruption, labeled cells in the tooth-half of the periodontal ligament moved incisally at a rate similar to the eruption rate. In the pinned incisors, no further incisal migration could be established. It is concluded that fibroblast migration in the tooth-half of the ligament is strictly coupled to the eruptive process.

T cell differentiation within thymic nurse cells.

Thymic nurse cells (TNC), defined as in vitro isolation products of thymic tissue, are epithelial cells harboring in their cytoplasm up to 200 intact, actively dividing thymocytes which are completely surrounded by vacuolar membranes. The TNC plasma membrane expresses major histocompatibility complex class I (H-2 K/D) and class II (I-A) antigens. The expression of MHC class I and class II antigens on the TNC vacuolar membranes was investigated with an improved in situ labeling technique. The major histocompatibility complex phenotype of the vacuolar membranes is H-2 K/D+, I-A2+ and thus identical to the TNC plasma membrane phenotype. By using the labeling technique, the TNC thymocyte population was examined for expression of the T cell differentiation antigens Thy-1, peanut agglutinin, Lyt-1, and Lyt-2, and the antigen expression was related to resistance of this population to cortisone. The majority of TNC thymocytes in individual TNC were cortisone-sensitive and expressed the immature phenotype of cortical thymocytes (Thy-1hi, PNAhi, Lyt-1lo, Lyt-2). A minority of the TNC thymocytes were cortisone-resistant and expressed a mature phenotype (Thy-1lo, peanut agglutininlo, Lyt-1hi). The existence of this minor mature population was confirmed in vivo: cortisone-resistant thymocytes were associated with cortical epithelial cells scattered throughout the thymic cortex of mice treated with dexamethasone. The major histocompatibility complex positive microenvironment of TNC and the heterogeneity in phenotype and resistance to cortisone of the TNC thymocytes, which is related to the state of maturation, indicate that TNC play an important role in the selection and differentiation of T cells.

T-cell differentiation in the rabbit. II. Con A and PHA response of thymus, spleen and lymph node cells and of thymus subpopulations; influence of dexamethasone treatment.

Mitogen-induced proliferation of rabbit lymphocytes from the thymus (Thy), spleen (Spl) and lymph node (LN) was measured by [3H]thymidine incorporation. The various cell suspensions taken from normal animals showed wide differences in their response to Con A and PHA. Cell suspensions taken from steroid-treated animals did not show differences in response. The disappearance of these differences in mitogen reactivity after steroid treatment is caused by a decreased Con A response of LN cells and an increased Con A and PHA response of Thy cells. The results suggest the existence of a steroid-sensitive (Ss). Con A-responsive cell population in LN cells. The mitogen response of untreated and steroid-resistant (SR) Thy cells was further investigated in cell suspensions separated on density gradients and in cell suspensions enriched in, or depleted of cells bearing receptors for Fc (T gamma and T non-gamma, respectively). It is concluded that Ss thymocytes of high density are mitogen non-reactive, and that Ss cells of low and medium density consist of subpopulations of cells reactive to Con A and/or to PHA. Sr thymocytes still displayed heterogeneity in buoyant density and mitogen responsiveness. Heavy cells and T gamma cells (10% of the Sr cells were T gamma) showed a lowered mitogen response. The results are discussed in relation to data describing the localization of the cell types which differ in mitogen reactivity. The results support the idea of two differentiation pathways, one for Ss and the other for Sr thymocytes.