Comparison of growth and metabolism of avian osteoblasts on polished disks versus thin films of titanium alloy.

The purpose of this study was to evaluate the efficacy of using high vacuum, thermal evaporation to deposit thin films of Ti-6Al-4V onto plates for subsequent cell culture investigations. Osteoblastic response to thin-film coated plates was compared to that of cells grown on Ti alloy disk inserts and uncoated culture plates. The Ti alloy disks were polished, cleaned, and passivated following a commercial protocol for orthopedic implants. Mean surface roughness was 262 nm for the Ti alloy disks and 4.756 nm for the coated culture plates. Osteoblasts isolated from 16-day chick embryo calvariae were cultured on polystyrene, thin films, and disks. At confluence, the cells were cultured an additional 48 h and were evaluated for cell number (DNA content), rate of glycolysis (lactate production), alkaline phosphatase activity (ALPase), and collagenous (3H-proline hydroxylation) and noncollagenous protein synthesis. Cell morphology was similar for the controls, disks, and thin-film groups. DNA, lactate, cell layer ALPase, 3H-hydroxyproline, and noncollagenous protein were not different (p > 0.05) among the control, thin-film, and disk groups. Medium ALPase was lower (p < 0.05) in the thin-film group compared to the control group. Although aluminum and vanadium percentages varied from nominal in the thin-film groups (11Al-2V as opposed to 6Al-4V), avian osteoblasts responded similarly to the Ti alloy thin films, disks, and uncoated culture plates for the smooth surfaces tested. The thin-film cell culture system used for elemental material studies appears to offer a promising method for the investigation of cellular response to alloyed biomaterials as well. Proper adjustments in alloy percentages before deposition, however, need to be made if thermal evaporation is utilized.

Hydrogen peroxide inhibits giant cell tumor and osteoblast metabolism in vitro.

This study investigates the efficacy of using hydrogen peroxide as adjuvant therapy after extended local curettage for benign giant cell tumors of bone. Hydrogen peroxide is used clinically as a chemical adjuvant for removal of residual tumor cells, presumably by effervescent cleansing with minimal damage to surrounding soft tissue and bone cells. This investigation examined the effects of hydrogen peroxide on giant cell tumor cells and osteoblasts grown in culture. Fresh fragments of histologically confirmed giant cell tumor tissue (six patients) and trabecular bone (one patient) were excised. Cells obtained from the fragments were grown in culture. Confluent cell cultures were exposed to saline (control) or hydrogen peroxide (0.1-1000 mm) for 2 minutes, and incubation was continued for 12, 24, or 48 hours without hydrogen peroxide. Protein content, deoxyribonucleic acid content, tartrate resistant acid phosphatase activity, and alkaline phosphatase activity were measured in the cell layers. The medium from the final 12 hours of each incubation period was used to evaluate lactate production. Cell lysis or death occurred after exposing giant cell tumor cells and osteoblasts to 100 mm and 30 mm hydrogen peroxide, respectively, concentrations substantially lower than the 3% (880 mm) hydrogen peroxide commonly used clinically. These results support the theory of using a minimal concentration of hydrogen peroxide as a chemical adjuvant in the surgical treatment of giant cell tumors of bone.

Extracellular pH modulates the activity of cultured human osteoblasts.

The effect of medium pH on the activity of cultured human osteoblasts was investigated in this study. Osteoblasts derived from explants of human trabecular bone were grown to confluence and subcultured. The first-pass cells were incubated in Hepes-buffered media at initial pHs adjusted from 7.0 to 7.8. Osteoblast function was evaluated by measuring lactate production, alkaline phosphatase activity, proline hydroxylation, DNA content, and thymidine incorporation. Changes in medium pH were determined from media pHs recorded at the beginning and end of the final 48 h incubation period. As medium pH increased through pH 7.6, collagen synthesis, alkaline phosphatase activity, and thymidine incorporation increased. DNA content increased from pH 7.0 to 7.2, plateaued from pH 7.2 to 7.6, and increased again from pH 7.6 to 7.8. The changes in the medium pH were greatest at pHs 7.0 and 7.8, modest at pHs 7.4 and 7.6, and did not change at 7.2, suggesting that the pHs are migrating towards pH 7.2. Lactate production increased at pH 7.0 but remained constant from 7.2 to 7.8. These results suggest that in the pH range from 7.0-7.6 the activity of human osteoblasts increases with increasing pH, that this increase in activity does not require an increase in glycolytic activity, and that pH 7.2 may be the optimal pH for these cells.

Comparison of human osteoblasts and osteogenic cells from heterotopic bone.

Heterotopic ossification is a common complication in which bone develops in soft tissues. Although frequently benign, in some patients the condition becomes painful, restricts motion, and requires surgical intervention. This condition and the cells responsible for it are poorly characterized. Using cell culture techniques, this study compares the performance of osteogenic cells obtained from heterotopic ossification with trabecular osteoblasts isolated from the same patient. Cells isolated from trabecular bone samples and heterotopic ossification sites from six patients were evaluated for osteocalcin production after exposure to 1,25-dihydroxycholecalciferol, alkaline phosphatase activity, typing and synthesis of collagen, cell proliferation, and total protein content. Samples of heterotopic ossification and trabecular bone from three of the patients were examined histologically. Heterotopic ossification derived cells were shown to produce osteocalcin, Type 1 collagen, and alkaline phosphatase activity. They also had increased rates of collagen synthesis, alkaline phosphatase activity, and cell proliferation compared with the normal osteoblasts. Initial tissue from the heterotopic ossification sites showed increased numbers of osteocytes/mm2 compared with normal trabecular bone. Although heterotopic ossification derived cells functioned qualitatively like osteoblasts, they exhibited elevated levels of activities traditionally ascribed to osteoblasts, such as collagen synthesis and alkaline phosphatase activity.

Toxic effects of wound irrigation solutions on cultured tibiae and osteoblasts.

Irrigating wounds with solutions of antiseptic or antibiotic agents is routinely performed in orthopaedic surgery to reduce the incidence of microbial infection. The effects of these agents on healthy bone tissue is unknown. Three commonly employed antiseptic agents (hydrogen peroxide, Betadine solution, Betadine scrub) and one antibiotic solution (bacitracin) were tested on tibiae and osteoblasts isolated from embryonic chicks. Osteoblast function was evaluated by glycolytic energy metabolism (lactate production), cell number (DNA content), and collagen synthesis ([3H]proline hydroxylation). Two series of experiments were performed. To study concentration-related effects, tibiae or cells were exposed to a range of concentrations of the agents (diluted in saline, 0.9% NaCl) for 2 min, rinsed with saline, and incubated for 24 h in medium containing [3H]proline. For the recovery study, the cells were exposed to an effective, but nonlethal, concentration of the antiseptic agents for 2 min, rinsed with saline, and the incubation was continued in complete culture medium for 6, 12, 24, 48, or 72 h with [3H]proline added for the final 6 h. Solutions containing the antiseptic agents were cytotoxic to both bones and cells at concentrations well below those used clinically in irrigation solutions. In contrast, bacitracin at the concentrations tested was safe for osteoblasts and tibiae. These results suggest that the use of irrigation solutions containing H2O2, Betadine solution, or Betadine scrub on exposed bone tissue should be considered with caution.

Quantitative analysis of 2-oxoglutarate in biological samples using liquid chromatography with electrochemical detection.

This paper describes a technique for quantitative analysis of 2-oxoglutarate (alpha-ketoglutarate) in biological samples using liquid chromatography with electrochemical detection (LC-EC). This method utilizes a simplified, efficient sample preparation designed to select for 2-oxoglutarate and similar compounds by derivatization with phenylhydrazine. The response was linear over the range from 62.5 to 1000 ng/ml. The least quantifiable concentration was 62.5 ng/ml and the least detectable concentration was 25 ng/ml. To test the ability of the assay to measure 2-oxoglutarate in biological samples, this method was used to quantitate the 2-oxoglutarate content in chick osteoblast cultures and to determine the ability of the assay to accurately measure a standard addition of 500 ng/ml 2-oxoglutarate when added to a sample of the forementioned groups. The 2-oxoglutarate content of these cells was 6.67 +/- 1.20 ng/micrograms DNA or 105 +/- 18 ng/cell layer (mean +/- 95% confidence interval) and the assay accurately measured the standard addition. This method was also used to quantitate 2-oxoglutarate content in whole embryonic chick calvariae containing 6.40 +/- 0.95 ng/mg dry bone weight or 37.5 +/- 5.5 ng/bone. This assay provides significantly lower detection limits than the currently available procedures and is suitable for determination of 2-oxoglutarate in biological samples where very low amounts of 2-oxoglutarate are found. This method is the first application of LC-EC for quantitating 2-oxoglutarate.

Medium pH modulates matrix, mineral, and energy metabolism in cultured chick bones and osteoblast-like cells.

The effects of medium pH were tested on calvariae, tibiae, and osteoblast-like cells from chick embryos. Bones and isolated cells were incubated for 5 h or 2 days in Hepes-buffered medium at pH values ranging from 6.8 to 8.2. Osteoblast function was evaluated by lactate production, oxygen consumption, alkaline phosphatase activity (AlPase), Ca and inorganic phosphate (Pi) flux, proline hydroxylation, DNA content, and thymidine incorporation. As medium pH was increased, glycolysis, collagen synthesis, and AlPase increased, while Ca efflux decreased. No effect of pH was seen on mitochondrial activity, Pi efflux, or cell number or proliferation. The importance of glycolysis as an endogenous pH regulator was demonstrated by inhibition with iodoacetic acid or glucose restriction and by adding lactate to the medium. The results suggest that the pH of bone interstitial fluid may be regulated by glycolysis and that changes in pH of this compartment may have marked effects on osteoblast function.

Topically active ocular carbonic anhydrase inhibitors: novel biscarbonylamidothiadiazole sulfonamides as ocular hypotensive agents.

A novel homologous series of bis(carbonyl)amidothiadiazole sulfonamides has been synthesized for structure-activity relationship studies, and initial characterization has been performed. The goal was synthesis of thiadiazole derivatives with appropriate lipid and water solubilities for utility as topically (corneal application) active carbonic anhydrase (CA) inhibitors. This series has solubility properties and pKa which bracket those of acetazolamide--the prototypical CA inhibitor. All of these compounds are active as in vitro CA inhibitors, and are 10-25% as potent as acetazolamide as in vitro enzyme inhibitors. Two of these compounds act as ocular hypotensive agents after topical application of a single dose to the corneas of normotensive New Zealand albino rabbits. The efficacy of the lead compound of this series (in this one model) is approximately equivalent to that of topical CA inhibitors that are presently in clinical trial. None of these novel compounds reacts to an appreciable extent with free sulfhydryl groups (a predictor of toxicity). This family of compounds will be useful for future studies of ocular pharmacokinetics, as well as ocular and systemic effects of topical administration of CA inhibitors. These and future studies may lead to development of thiadiazole sulfonamides useful in the management of glaucoma.