chondroitin-sulfates and fluorexon

Osteoclasts are involved in bone resorption, and its activation is considered one of the causes of osteoporosis. The pit assay is the principal method for evaluating osteoclast function by measuring hydroxyapatite resorption in vitro. However, the pit assay requires time and trained techniques, including the pit image analysis, and there is no other easy method for evaluating bone resorption. In this study, we developed a novel approach to quantify the bone resorption activity using a calcium phosphate (CaP) coating labeled with fluorescent polyanion. Fluoresceinamine-labeled chondroitin polysulfate or Hoechst 33258-labeled deoxyribonucleic acid was used for CaP labeling. When macrophage cell line RAW264 was cultured on the labeled CaP under the stimulation with the receptor activator of the NF-κB ligand (RANKL), RAW264 cells differentiated into osteoclastic cells and the fluorescence intensity of the culture supernatant and pit area increased in a time- and dose-dependent manner. Furthermore, drugs for osteoporosis treatment, such as pamidronate and β-estradiol, inhibited fluorescein release by the cells stimulated with RANKL. A positive correlation between the fluorescence intensity and pit area was observed (r=0.917). These results indicated that this new method using fluorescent polyanion-labeled CaP is a standardized useful assay system for the evaluation of bone resorption activity.

Topics: Animals; Bisbenzimidazole; Bone Resorption; Calcium Phosphates; Cell Line; Chondroitin Sulfates; DNA; Drug Evaluation, Preclinical; Fluoresceins; Fluorescent Dyes; Mice; Osteoclasts; Osteoporosis; Polymers

To improve corneal endothelial cell survival during cold preservation by the addition of a compound that enhances cell membrane repair.. Cell survival was assessed using intracellular accumulation of the fluorescent molecule calcein as a marker for endothelial cell viability in bovine corneas stored at 4 degrees C in Optisol-GS. The effect on endothelial cell survival of artificially lowering the surface tension of the cell membrane was also assayed by the addition of a surfactant, Poloxamer 188 (PLURONIC F68), which has been shown to reseal damaged cell membranes, even under conditions where metabolism is inhibited.. Endothelial cell survival in corneas stored at 4 degrees C in Optisol-GS was increased by the addition of 1 mg/mL Poloxamer 188. The level of dead or missing corneal endothelial cells was less than 1% for corneas stored for 6, 9, or 12 days in Optisol-GS with 1 mg/mL Poloxamer 188 at 4 degrees C. In contrast, dead or missing corneal endothelial cell percentages were increased to approximately 4% after 9 days and approximately 10% after 12 days in storage in Optisol-GS without Poloxamer 188 at 4 degrees C.. The surfactant Poloxamer 188 enhances endothelial cell survival in cold-stored bovine corneas.

Topics: Animals; Cattle; Cell Count; Cell Survival; Chondroitin Sulfates; Cold Temperature; Complex Mixtures; Culture Media, Serum-Free; Dextrans; Endothelium, Corneal; Fluoresceins; Fluorescent Dyes; Gentamicins; Microscopy, Fluorescence; Organ Preservation Solutions; Poloxamer; Surface-Active Agents