acid-phosphatase and calcium-phosphate--dibasic--anhydrous

Equine osteoclast-like cells (OCLs) were generated from the bone marrow (BM) of two ponies and one horse in the presence of RANKL, the receptor activator of NF kappa B ligand and macrophage colony-stimulating factor (M-CSF). The phenotype of these cells was confirmed by demonstration of characteristics typical of osteoclasts (OCs) including: the expression of tartrate-resistant acid phosphatase (TRAP), the vitronectin receptor (VNR) and the calcitonin receptor (CTR), the demonstration of responsiveness to calcitonin (CT) and the ability to form resorption lacunae on ivory slices and calcium phosphate films. The bisphosphonate pamidronate (APD) dose-dependently inhibited resorption of calcium phosphate films by equine OCLs with an IC(50) of 5.8 x 10(-7) M in one horse. APD also dose-dependently inhibited the number of OCLs present in BM cultures after 7 days. However, this effect is most likely attributable to increased OCL death rather than decreased OCL formation. Paradoxically, ADP appeared to cause an early, transient, increase in OCL formation in BM cultures, however, this effect was reversed after 7 days. These preliminary in vitro data support the potential use of APD in clinical conditions characterised by increased bone turnover such as osteomyelitis, osteitis, septic osteoarthritis, navicular disease, cystic bone lesions and immobilisation-induced osteoporosis and provide useful information for future pharmacokinetic studies and clinical trials in vivo.

Topics: Acid Phosphatase; Animals; Bone Marrow; Bone Resorption; Calcium Phosphates; Cell Death; Cells, Cultured; Diphosphonates; Dose-Response Relationship, Drug; Gene Expression Regulation; Horses; Inhibitory Concentration 50; Iodine Radioisotopes; Osteoclasts; Osteogenesis; Pamidronate; Receptors, Vitronectin; Staining and Labeling; Time Factors

The use of calcium phosphate biomaterials as a bone substitute necessitates the use of normative biocompatibility and biodegradation techniques which must be fast, simple and reproducible. In the present study, we have developed an in vitro model to study and to compare different calcium phosphate ceramics. After activation with 1,25-dihydroxy-vitamin D3 and phorbol 12,13-dibutyrate, the monoblastic U937 cells became multinucleated, expressed tartrate-resistant acid phosphatase and several markers of monocyte/macrophage differentiation. Activated U937 cells did not express the vitronectin receptor (VNR) (as revealed using monoclonal antibodies 23C6 or 13C2) but around 25% of the cells were strongly reactive with 211D, a novel monoclonal antibody that recognizes an osteoclast-specific membrane antigenic determinant. These cells remain active/viable with hydroxyapatite (HA) or beta-tricalcium phosphate (beta-TCP) ceramics. In conclusion, activated U937 cells are good candidates to use in a normative in vitro method to evaluate new biomaterials.

Topics: Acid Phosphatase; Antibodies, Monoclonal; Antibody Specificity; Biocompatible Materials; Biodegradation, Environmental; Calcitriol; Calcium Phosphates; Ceramics; Dentin; Flow Cytometry; Giant Cells; Humans; Integrins; Leukemia, Myeloid; Macrophages; Microscopy, Electron, Scanning; Monocytes; Phorbol 12,13-Dibutyrate; Receptors, Cytoadhesin; Receptors, Vitronectin; Reproducibility of Results; Staining and Labeling; Tumor Cells, Cultured

The efficiency of DNA transfection into mammalian cell cultures has been monitored using a variety of reporter assays. However, the common procedures are expensive, time-consuming and usually cannot identify the transfected cell population directly. In the present communication we describe a simple, inexpensive and efficient method to directly identify DNA transfection in mammalian cells using tartrate-resistant acid phosphatase (TRAP) gene expression. The method involves the transfection of a plasmid (pCT3), which contains TRAP cDNA driven by a CMV promoter, into mammalian cells. The cells can then be stained for TRAP activity, and the transfection efficiency can be determined by simply counting the positively transfected cells in a defined area with a microscope. This method permits screening of mammalian cells for transfection efficiency in multi-well plates. After waiting 30-40 minutes to allow the TRAP assay to saturate, wells can be scored in 1-2 minutes with little difficulty in detecting the transfected cells.

Topics: Acid Phosphatase; Animals; beta-Galactosidase; Calcium Phosphates; Cells, Cultured; Cloning, Molecular; Cytomegalovirus; DNA, Complementary; Gene Expression; Humans; Promoter Regions, Genetic; Staining and Labeling; Tartrates; Transfection

Hydroxyapatite (HA) and tricalcium phosphate (TCP) are useful for grafting and augmentation of bone tissue. In this study, conventional and histochemical transmission electron microscopy were used to study osteogenic events at the interface between the implanted materials and adjacent tissue from 1 to 4 weeks postoperatively. The microscopic results indicated that TCP was resorbed more rapidly than HA after implantation, with a notable breakdown of material and replacement by mesenchymal cells with ultrastructural features resembling osteoprogenitor cells and collagen up to 4 weeks postoperatively. Alkaline phosphatase and acid phosphatase reactivity in the tissues helped to identify and differentiate the histologic differences observed between HA and TCP.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Bone and Bones; Calcium Phosphates; Histocytochemistry; Hydroxyapatites; Male; Microscopy, Electron; Osteogenesis; Rats; Rats, Wistar

Dicalcium phosphate dihydrate, CaHPO4 . 2H2O (DCPD) crystals were grown in solutions and in different types of gels (silica, agar, collagen) at different pH's, in the presence of other ions, and on different Ca-bearing substrates (enamel, dentine, bone, calcite). Hydrolysis of DCPD to other calcium phosphates and calcium oxalates were made in solutions containing different ions at 37 degrees C, 24h. Results showed that the type of media (solution or gel), type of gel, pH, presence of other ions affect the morphology (size/shape) and growth of DCPD. In systems of changing pH, the more basic calcium phosphate (e.g., apatite, OCP) formed in the first zone, DCPD in the last zone, regardless of the initial Ca/P of the system. Growth of DCPD was suppressed in the presence of F-(which favored growth of apatite), of Cd2+, Pb2+ (which favored OCP). Hydrolysis of DCPD to apatite was promoted in solutions containing Ca2+, F-, CO3=, and inhibited by Mg2+, P2O74-. Hydrolysis of DCPD to calcium oxalate occurs in presence of C2O4=. Results indicate that the variation in morphology of DCPD occurring in various human tissue calcinosis and the co-existence of DCPD with other calcium phosphates and oxalates in urinary and dental calculi are due to variations in pH, and presence of different ions in the media.

Topics: Acid Phosphatase; Animals; Bone and Bones; Cadmium; Calcium Phosphates; Chemical Phenomena; Chemistry; Crystallization; Dental Enamel; Fluorides; Humans; Lead; Sharks; X-Ray Diffraction