Tartrate-resistant Acid Phosphatase Reaction Research Articles

Effect of The Receptor Activator of Nuclear Factor кB and RANK Ligand on In Vitro Differentiation of Cord Blood CD133(+) Hematopoietic Stem Cells to Osteoclasts.

Receptor activator of nuclear factor-kappa B ligand (RANKL) appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells (HSCs). This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor (CTR) on cord blood HSC surface. In this experimental study, CD133(+) hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase (TRAP) staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction (RT-PCR) assay for specific genes. Hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells. Presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast.

The Effect of Etidronate on the Periodontium of Ovariectomized Rats

Bisphosphonates are indicated for the treatment of osteoporosis. However, they could have an adverse effect on specific sites, such as the bisphosphonate-related osteonecrosis of the jaw. The aim of this study is to investigate the effect of etidronate on the resorption and apposition sides of the periodontium in ovariectomized rats. Twenty-four female Wistar rats were randomly subjected to either ovariectomy or sham operation. After 8 weeks, six animals of each group were sacrificed. The other 12 rats received 5 mg/kg/day etidronate subcutaneously during 4 weeks. Tartrate-resistant acid phosphatase reaction and immunohistochemical staining for receptor activator of nuclear factor-κB (RANK), RANK-ligand (RANKL), osteoprotegerin (OPG), and osteocalcin was performed. Immunoreactivity was evaluated using a semiquantitative analysis. In ovariectomized rats, osteoclasts were noticed in the root socket of molars, including the apposition side of the periodontium, in which RANKL expression was significantly evidenced. In the etidronate-treated group, OPG expression was significantly expressed and osteoclasts that were noticed in the resorption side remained undetected in the apposition side even under ovariectomy. RANK was significantly expressed in ovariectomized rats treated with etidronate. Osteoid formation and osteocalcin expression were described on the alveolar bone surfaces in etidronate-treated rats, with or without ovariectomy. Etidronate has specific site and bone cell actions in the periodontium. It inhibits the osteoclast differentiation induced by ovariectomy in the apposition side of the periodontium but maintains bone formation over all the socket surfaces. Such specificity may be related to the pathogenesis of the bisphosphonate-induced osteonecrosis of the jaw.

Eccentric Localization of Osteocytes Expressing Enzymatic Activities, Protein, and mRNA Signals for Type 5 Tartrate-resistant Acid Phosphatase (TRAP)

Enzymatic activity of type 5 tartrate-resistant acid phosphatase (TRAP) has been regarded as one of the reliable markers for osteoclasts and their precursors. The presence of TRAP activity in osteocytes near the bone resorbing surface has also been pointed out in some reports. However, the significance of TRAP reactions in osteocytes remains controversial and, in fact, there is no agreement as to whether the histochemical enzyme reactions in osteocytes represent the TRAP enzyme generated by the respective osteocytes or is a mere diffusion artifact of the reaction products derived from the nearby osteoclasts. Current histochemical, immunohistochemical, and in situ hybridization studies of rat and canine bones confirmed TRAP enzyme activity, TRAP immunoreactivity, and the expression of Trap mRNA signals in osteocytes located close to the bone-resorbing surface. TRAP/Trap- positive osteocytes thus identified were confined to the areas no further than 200 microm from the bone-resorbing surface and showed apparent upregulation of TRAP/Trap expression toward the active osteoclasts. Spatial and temporal patterns of TRAP/Trap expression in the osteocytes should serve as a valuable parameter for further analyses of biological interactions between the osteocytes and the osteoclasts associated with bone remodeling.

Combined TUNEL and TRAP methods suggest that apoptotic bone cells are inside vacuoles of alveolar bone osteoclasts in young rats.

Although it is generally accepted that osteoclasts breakdown and resorb bone matrix, the possibility that they may also be able to engulf apoptotic osteoblasts/lining cells and/or osteocytes remains controversial. Apoptosis of osteoblasts/ lining cells and/or osteocytes and interactions between these cells and osteoclasts are extremely rapid events that are difficult to observe in vivo. A suitable in vivo model for studying these events is the alveolar bone of young rats because it is continuously undergoing intense resorption/remodeling. Thus, sections of aldehyde fixed alveolar bone of young rats were stained by the combined terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and the tartrate-resistant acid phosphatase (TRAP) method for the simultaneous visualization of apoptotic cells and osteoclasts in the same section. The combined TUNEL and TRAP reactions, in the same section, greatly facilitated visualization of relationship between osteoclasts and apoptotic bone cells during alveolar bone remodeling. Our results showed that several TRAP-positive osteoclasts exhibited large vacuoles containing TUNEL positive apoptotic structures, probably derived from osteoblasts/lining cells and/or osteocytes. These results support the idea that alveolar bone osteoclasts are able to internalize dying apoptotic bone cells.

Methods for the differentiation of giant cells in canine and feline neoplasias in paraffin sections.

In the following study cells with at least two cell nuclei are addressed as giant cells. In 47 biopsies of feline neoplasias (fibrosarcoma, haemangioendothelsarcoma, mammary adenocarcinoma, osteoidsarcoma, complex sarcoma), and 25 biopsies of canine neoplasias (malignant seminoma, mammary adenocarcinoma, haemangioendothelsarcoma, fibrosarcoma, osteoblastic sarcoma, complex sarcoma) giant cells are distinguished either as neoplastic giant cells or as reactive (non-neoplastic) giant cells. Cell nuclei of neoplastic giant cells which are labelled with the monoclonal antibody MIB 1 are mitotic active; cell nuclei are polymorph and can show atypical mitosis; the cytoplasmic reaction with tartrate resistant acid phosphatase (TRAP) is negative. Negative reactions with MIB 1, positive TRAP staining and homogeneous cell nuclei are distinctive for osteoclast-like glant cells. Other non-neoplastic giant cells (e.g. foreign body cells, Langhans-giant cells) are negative with both MIB 1 and TRAP. Double staining of paraffin sections is possible. Routine formalin-fixation, embedding in paraffin and decalcifying tissue samples do not interfere with MIB 1 immunoreactions or TRAP reactions. Methodological modifications that were necessary for the preparation of paraffin sections from canine and feline tissue samples are discussed. As the presence of neoplastic giant tumour cells is an index for a poor prognosis in human medicine, not only the entity of the tumour must be named, but also the exact significance of the giant cell type:, e.g. fibrosarcoma with osteoclast-like giant cells, hepatic carcinoma with reactive giant cells, malignant seminoma with neoplastic giant cells, angiosarcoma with both neoplastic giant cells and osteoclast-like giant cells. This would enable the classification of further neoplasias dealing with clinical courses of the diseases. Over the past years our stains have remained stable. It is possible to carry out retrospective investigations with archived tissue samples and make permanent preparations. A reclassification and a refined form of diagnosis (tumour and giant cell type) would be recommended.

Establishment of a new cell line from a patient with hairy cell leukemia-japanese variant

A cell line, JHC-2, was established from the peripheral blood of a patient with hairy cell leukemia (HCL)-Japanese variant. The JHC-2 cells have cytologic features similar to those of the original tumor cells. They displayed hairy cytoplasmic projections by phase contrast and scanning electron microscopy. The tartrate-resistant acid phosphatase reaction was weakly positive. The immunophenotype of the JHC-2 cells was CD5–, CD10–, CD11c±, CD19+, CD21+, CD23+, CD24–, CD25± CD38– and FMC-7+. The expression of surface immunoglobulin (IgG, k) and the configuration of Ig gene rearrangements in the JHC-2 cells were identical to those in the original leukemic cells, and the JHC-2 cells displayed trisomy 9 on cytogenetic examination. Southern blot analysis for the Epstein-Barr virus (EBV) genome showed that the JHC-2 cells contained the EBV genome, although the freshly isolated leukemic cells did not. These results indicate that the JHC-2 cell line is an EBV spontaneously transformed B cell line originating from HCL cells.

Topography of forming and resorbing cells on endosteal surfaces of the rabbit humerus by double-staining with in situ hybridization and tartrate-resistant acid phosphatase reaction: a new model to study the bone reaction to loading

Since the first investigations made by Wolff, it has been known that bone adapts to mechanical load. The mechanisms which guide the reaction of osteoblasts and osteoclasts to load are still insufficiently known. In situ hybridization (ISH) allows the detection of intracellular gene transcripts. Therefore, the ISH technique was further developed to allow the detection of pro-alpha 1 (I) collagen gene transcripts on undecalcified bone surfaces. Additionally, this new technique was combined with the tartrate resistant acid phosphatase technique. The combination of the two methods allows the detection of forming and resorbing cells on the same undecalcified bone surface. In addition, a new animal model was developed to study the reaction of bone to mechanical load. This model mimics the situation of bone implants (e.g. hip prosthese), which is a static situation which is dynamically loaded by the action of the patient.

Morphological and histochemical comparison of the cells elicited by ectopic bone implants and tibial osteoclasts

Pellets of mineralized and demineralized bone and a composite mixture of mineralized and demineralized, devitalized bone particles were implanted subcutaneously on the dorsal body wall of young adult rats. Two weeks post-implantation, the pellets were removed and processed for histochemical and morphological analyses. Rat proximal tibia was also processed for evaluation. The levels of tartrate-resistant acid phosphatase (TRAP) activity in the multinucleated giant cells (MNGCs) from each of the three implants and from osteoclasts were assessed using an image analyzer. The osteoclasts from the proximal tibia and the majority of MNGCs from the demineralized implants demonstrated high levels of TRAP activity. MNGCs from the mineralized implants showed either a low level or absence of TRAP activity. Most MNGCs from the composite implants exhibited a low level of TRAP activity; however, there was a population of cells that demonstrated a high level of reaction product, similar to that seen in the tibia and demineralized implant. Morphologically, osteoclasts from the proximal tibia and from the osteogenic demineralized implant exhibited ruffled borders. A small population of MNGCs from the composite implant also revealed osteoclastic features. In summary, MNGCs from the mineralized implant did not exhibit a level of TRAP reaction product or morphology similar to osteoclasts, while the majority of cells from the demineralized implant and a subpopulation of the MNGCs elicited by the composite implant did demonstrate TRAP expression and morphology similar to osteoclasts. The expression of osteoclastic characteristics in cells at an ectopic site may be dependent on accessory signals from the skeletal microenvironment; such signals appear to be absent from or incomplete in the mineralized implants but appear to be present when demineralized bone particles are implanted.

Quantification of Tartrate Resistant Acid Phosphatase Activity Using a Computerized Image Analysis System

Tartrate resistant acid phosphatase (TRAP) has been accepted as a marker for identification of osteoclasts. A method is reported here for quantitating TRAP using an image analysis system. The amount of the enzyme specific to osteoclasts can be used to differentiate osteoclasts from other cells capable of TRAP expression. TRAP expression characteristic of the osteoclast was compared with that of multi-nucleated giant cells (MNGC)s recruited to the site of subcutaneously implanted mineralized bone matrix. Two weeks post-implantation, the pellets were removed and processed for the demonstration of TRAP along with rat proximal tibiae. A large amount of TRAP was consistently expressed by the in situ osteoclasts. The MNGCs associated with the mineralized bone implants expressed little if any TRAP reaction product. Using this system, the amount of TRAP reaction product or any other enzyme reaction product expressed can be objectively and reproducibly quantitated.

Chondroclasts in osteoneogenesis

Allogenic, demineralized bone powder (DBP) was implanted into rat rectus abdominis muscle to induce Osteoneogenesis. The main induction steps are invasion of the implant by host mesenchyme cells, differentiation of cartilage, invasion by blood capillaries or angiogenesis, differentiation of osteoblasts and bone marrow. The result is the formation of a cancellous ossicle. Giant polykarions appear in the implant after calcification of the cartilage matrix. As the DBP particles are not resorbed in the implant, these polykarions could either be foreign body giant cells brought about in reaction to foreign matrix or chondroclasts which resorb the cartilage. The results obtained by histological and histochemical methods (McNealsvon Kossa stain, tartrate resistant acid phosphatase reaction), as well as ultrastructural studies, lead to the conclusion that these large polynucleated cells are chondroclasts.

Severe Anaemia with Unusual Blasts in the Bone Marrow

This Quiz refers to an iliac crest biopsy performed in a 32-year-old man suffering from haemolytic anaemia. Deficiency of glucose 6-phosphate dehydrogenase was known in this patient for many years. Due to an acute haemolytic crisis accompanied by pancytopenia he had to be hospitalised. The clinical examination revealed a haemoglobin concentration drastically decreased to 5g/dl, whereas the reticulocyte count was reported to be slightly subnormal with an actual value of 1.2%. The blood concentration for lactate dehydrogenase was significantly increased up to 500 D/ml: Leucocytes (2.3 X 109 1) and platelets (87 x 109 1) were both decreased in number. The trephine biopsy taken from the iliac crest consisted of a cylinder of cancellous bone measuring 30 mm in length. The specimen was fixed in a mixture of glutaraldehyde (0.5% v/v), formaldehyde (1.2% v/v) and calcium acetate (1.85% w/v), subsequently decalcified in neutral EDTA and embedded in paraffin. Sections were stained with Giemsa, Gomori and PAS according to routine procedures, and for chloroacetate esterase and tartrate resistant acid phosphatase1. The cancellous bone did not show any signs of an enhanced turnover. By means of the tartrate resistant acid phosphatase reaction only very few inactive osteoclasts