Expression of endothelial nitric oxide synthase protein is not necessary for mechanical strain-induced nitric oxide production by cultured osteoblasts.

UNLABELLED: Regulation of nitric oxide (NO) production is considered essential in mechanical load-related osteogenesis. We examined whether osteoblast endothelial NO synthase (eNOS)-derived NO production was regulated by HSP90. We found that HSP90 is essential for strain-related NO release but appears to be independent of eNOS in cultured osteoblasts. INTRODUCTION: NO is a key regulator of bone mass, and its production by bone cells is regarded as essential in mechanical strain-related osteogenesis. We sought to identify whether bone cell NO production relied upon eNOS, considered to be the predominant NOS isoform in bone, and whether this was regulated by an HSP90-dependent mechanism. METHODS: Using primary rat long bone-derived osteoblasts, the ROS 17/2.8 cell line and primary mouse osteoblasts, derived from wild-type and eNOS-deficient (eNOS(-/-)) mice, we examined by immunoblotting the expression of eNOS using a range of well-characterised antibodies and extraction methods, measured NOS activity by monitoring the conversion of radiolabelled L-arginine to citrulline and examined the production of NO by bone cells subjected to mechanical strain application under various conditions. RESULTS: Our studies have revealed that eNOS protein and activity were both undetectable in osteoblast-like cells, that mechanical strain-induced NO production was retained in bone cells from eNOS-deficient mice, but that this strain-related induction of NO production was, however, dependent upon HSP90. CONCLUSIONS: Together, our studies indicate that HSP90 activity is essential for strain-related NO release by cultured osteoblasts and that this is highly likely to be achieved by an eNOS-independent mechanism.

Identifying the cellular basis for reimplantation failure in repair of the rotator cuff.

We examined cultured osteoblasts derived from paired samples from the greater tuberosity and acromion from eight patients with large chronic tears of the rotator cuff. We found that osteoblasts from the tuberosity had no apparent response to mechanical stimulation, whereas those derived from the acromion showed an increase in alkaline phosphatase activity and nitric oxide release which is normally a response of bone cells to mechanical strain. By contrast, we found that cells from both regions were able to respond to dexamethasone, a well-established promoter of osteoblastic differentiation, with the expected increase in alkaline phosphatase activity. Our findings indicate that the failure of repair of the rotator cuff may be due, at least in part, to a compromised capacity for mechanoadaptation within the greater tuberosity. It remains to be seen whether this apparent decrease in the sensitivity of bone cells to mechanical stimulation is the specific consequence of the reduced load-bearing history of the greater tuberosity in these patients.

NOS isoforms in adult human osteocytes: multiple pathways of NO regulation?

Until now, eNOS has been considered to be the predominant osteocytic nitric oxide synthase (NOS) isoform in bone. We previously studied the distribution of eNOS protein expression in the human femoral neck because of its possible involvement in the response to load. Studies in rat and human fracture callus have shown that nNOS mRNA is expressed sometime after fracture, but no study has yet immunolocalized NOS isoforms in mature adult human bone. In this study, we have examined the distribution of NOS isoforms in iliac osteocytes. Frozen sections (10 microm) were cut from transiliac biopsies from 8 female osteoporotic patients (range, 56-80 years) and from 7 female postmortem femoral neck biopsies (range, 65-90 years). Sections were incubated overnight in antiserum for eNOS, nNOS, or iNOS followed by peroxidase/VIP substrate detection. We used eNOS and iNOS antisera directed against the C-terminus. For nNOS, three different antisera were used, two binding to different C-terminal epitopes and one binding to N-terminal epitope. Sections were then incubated in propidium iodide or methyl green to detect all osteocytes. eNOS antibody was able to detect eNOS epitopes in osteocytes. All three nNOS antibodies detected nNOS epitopes in osteocytes, but those directed against the C-terminus had higher detection rates. iNOS was rarely seen. In the iliac crest, the percentage of osteocytes positive for nNOS was higher than that for eNOS (cortical: nNOS 84.04%, eNOS 61.78%, P < 0.05; cancellous: nNOS 82.33%, eNOS 65.21%, P < 0.05). In the femoral neck, the percentage of osteocytes positive for nNOS (60.98%) was also higher than that for eNOS (40.41%), although this difference was not statistically significant. In conclusion, both eNOS and nNOS isoforms are present in osteocytes in the iliac crest and femoral neck.

Patterns of osteocytic endothelial nitric oxide synthase expression in the femoral neck cortex: differences between cases of intracapsular hip fracture and controls.

Evidence indicates that extensive amalgamation of adjacent resorbing osteons is responsible for destroying the microstructural integrity of the femoral neck's inferior cortex in osteoporotic hip fracture. Such osteonal amalgamation is likely to involve a failure to limit excessive resorption, but its mechanistic basis remains enigmatic. Nitric oxide (NO) inhibits osteoclastic bone destruction, and in normal bone cells its generation by endothelial nitric oxide synthase (eNOS, the predominant bone isoform) is enhanced by mechanical stimuli and estrogen, which both protect against fracture. To determine whether eNOS expression in osteocytes reflects their proposed role in regulating remodeling, we have examined patterns of osteocyte eNOS immunolabeling in the femoral neck cortex of seven cases of hip fracture and seven controls (females aged 68-96 years). The density of eNOS+ cells (mm(-2)) was 53% lower in the inferior cortex of the fracture cases (p < 0.0004), but was similar in the superior cortex. eNOS+ osteocytes were, on average, 22% further from their nearest blood supply, than osteocytes in general (p < 0.0001) and the nearest eNOS+ osteocyte was 57% further from its nearest canal surface (p < 0.0001). This differential distribution of eNOS+ osteocytes was significantly more pronounced in the cortices of fracture cases (p < 0.0001). We conclude that the normal regional and osteonal pattern of eNOS expression by osteocytes is disrupted in hip fracture, particularly at sites that are loaded most by physical activity. These results suggest that eNOS+ osteocytes may normally act as sentinels confining resorption within single osteons. A reduction in their number, coupled to an increase in their remoteness from canal surfaces, may thus permit the irreversible merging of resorbing osteons, and thus contribute to the marked increase in the fragility of osteoporotic bone.

A study to evaluate primary dressings for the application of cultured keratinocytes.

Despite the recent improvements in cell culture and dermal regeneration methods, tissue engineering of skin has yet to receive widespread acceptance in the management of burn injuries. The reasons for this are complex and include not only the inherent costs of (particularly) setting up and running such a system but also the continuing difficulties in achieving successful engraftment of the neoepidermis. The latter has previously been addressed in a number of ways, including improving the recipient bed and using pre-confluent delivery systems to allow earlier application of cells to that wound bed. One area that has received little attention is that of the optimal wound dressing to use with this technology; the cells are very poorly attached at early time points, and, in this context, the traditional dressing of paraffin gauze has never been formally assessed in comparison with newer materials. Using a porcine acute wound chamber model, we performed a prospective randomised trial to assess four different wound dressings with reference to the amount of epidermal cover gained and the histological quality of the regenerated skin after 3 weeks. Out of the four materials tested, polyurethane foam (Allevyn) was superior histologically (although equal in take rate with paraffin gauze), whilst polythene sheet (Opsite) and silicone sheet were substantially inferior. We conclude that the traditional dressing used with this technology should be compared with polyurethane foam in a clinical trial. In the future, novel dressings should be formally tested against traditional methods before being adopted.

Stability of piperacillin sodium in the presence of tazobactam sodium in 5% dextrose and normal saline injections.

The stability of piperacillin sodium in the presence of tazobactam sodium in 5% dextrose and normal saline i.v. admixtures has been determined using a modified stability-indicating high performance liquid chromatography assay method reported in the literature. The solutions were stored at room and refrigerator temperatures in plastic bags. They remained clear throughout the study although the pH values decreased slightly during storage. The solutions were stable for 2 days at 25 degrees C and for 28 days at 5 degrees C. Tazobactam sodium appears to have a slight adverse effect on the stability of piperacillin sodium.

Stability of ciprofloxacin in 5% dextrose and normal saline injections.

The stability of ciprofloxacin in 5% dextrose and normal saline i.v. admixtures have been determined using a stability-indicating high performance liquid chromatographic assay method reported in the literature. The solutions were stored in plastic bags, a procedure being used in hospitals for in or out-patient therapy. The solutions were stable for at least 3 months when stored at room or refrigerator temperatures. They remained clear throughout the study and their pH values did not change. The expiry date recommended by the manufacturer appears to be very conservative from a chemical viewpoint.

Stability of metronidazole in solutions and suspensions.

Excellent stable oral liquid dosage forms of metronidazole can be prepared from tablets or powder using commercially available vehicle containing suspending agents. Liquid dosage forms (5 mg/ml) can be prepared without suspending agents. The four samples prepared (10 and 5 mg/ml) were stable for at least 90 days at room temperature. Their pH values and physical appearances did not change on storage. When metronidazole free base is prescribed it may not be substituted with the ester (metronidazole benzoate) as is common practice in the U.S.A. Metronidazole tablets with a labelled expired date of April 1986 still showed 99.3% potency when assayed in January 1993.

Stability of foscarnet sodium in 5% dextrose and 0.9% sodium chloride injections.

The stability of foscarnet sodium injection in 5% dextrose and 0.9% sodium chloride injections was determined using a stability-indicating assay reported in the literature. The solutions were stable for at least 35 days when stored at 25 degrees or 5 degrees C. They remained clear throughout the study and pH values did not change. The solutions were stored in plastic bags as used by patients at home.

Stability of metronidazole benzoate in suspensions.

A stability-indicating HPLC assay method has been developed to quantify metronidazole benzoate in suspensions. A study of the stabilities of two suspensions (16.0 mg/ml) of metronidazole benzoate in commercially available vehicles, Ora-Plus and a mixture of Ora-Plus and Ora-Sweet, showed that both suspensions were stable for at least 90 days at room temperature. The mobile phase required to elute metronidazole benzoate contained 40% acetonitrile versus approximately 10% for the free base. The solubility of the ester in water at 25 degrees C was found to be approximately 0.1 mg/ml versus 10 mg/ml for the free base. The ester did not hydrolyse significantly to the free base after storage for 8 h at 37 degrees C in simulated gastric fluid, and 5 h at 37 degrees C in simulated intestinal fluid. The bitter tasting metronidazole may not be satisfactorily substituted with its tasteless ester in the treatment of local gastrointestinal infections.

The development of oral liquid dosage forms of metronidazole.

The stability of eight samples of metronidazole in aqueous vehicles was studied using a modified HPLC assay method. The samples were prepared using either metronidazole powder (5 mg/ml), metronidazole hydrochloride injection powder (10 mg/ml), or metronidazole powdered tablets (10 mg/ml). The samples prepared using metronidazole hydrochloride powder were stable and clear for at least 133 days at 25 degrees C. Their pH values were between 1.8 and 2.0. The samples prepared from metronidazole powder had pH values close to neutral and were stable for at least 45 days at room temperature (25 degrees C). However, to keep the drug in solution, the concentration of metronidazole powder was reduced to 5 mg/ml. The powdered tablets did not yield good uniform suspensions.

Development of a stable oral liquid dosage form of spironolactone.

A clear, stable, oral liquid dosage form of spironolactone has been developed. Solubility profiles of spironolactone were obtained in several co-solvent blends. Using this data, a co-solvent blend containing polyethylene glycol 400 (30% v/v), propylene glycol (10% v/v), glycerin (10% v/v) and ethyl alcohol (10% v/v) was used to solubilize spironolactone at a concentration of 2 mg/ml. The final formulation contained sweetening agents (sucrose, saccharin sodium), flavours (cherry, sweet), a desensitizing agent (menthol), a dye (FD&C Red #40) and a preservative (benzoic acid) to incorporate the desired organoleptic and preservative properties. A phosphate buffer was used to maintain a pH value of 4.5 (pH of maximum stability as reported earlier) to minimize hydrolysis. The final dosage form was stable for at least 93 days at 40 degrees C (loss of potency less than 4%). According to FDA guidelines, a tentative expiration date of 2 years at 25 degrees C is justifiable.

Stability of captopril in some aqueous systems.

A stability-indicating high performance liquid chromatographic method has been proposed to quantify captopril. The method has been used to determine the stability of captopril in oral liquid dosage forms prepared from either commercially available tablets or powder. The dosage forms in water were more stable than when the vehicle was a syrup. Furthermore, the dosage form prepared using powder in water was more stable than when tablets were used. While the decomposition of captopril followed first-order equation when the dosage forms were prepared in syrup (in two of the three solutions studied), this equation was not followed when water was the vehicle. This is probably due to an uncontrolled factor, oxygen, because captopril is very sensitive to oxidation. Captopril solution prepared in water using tablets was stable for about 20 days when stored at 5 degrees C, and that prepared using powder in water was stable for about 27 days. One commercial syrup hastened the process of decomposition with an additional unidentified product of decomposition.

Stability of cefuroxime axetil in suspensions.

Cefuroxime axetil is a mixture of two equally active isomers (50% each). A stability-indicating high-pressure liquid chromatography method for the quantification of cefuroxime axetil has been developed. The method is accurate, precise and reproducible. The percentage, relative standard deviation based on six injections was 1.2. Using the developed method, the chemical stability of cefuroxime axetil suspensions has been determined in three vehicles containing sugar and two without sugar. The results varied widely in the vehicles without sugar (up to +/- 31%). In the viscous vehicles containing sugar, the variations in the results were only up to +/- 6%. The cefuroxime axetil in these suspensions was stable up to 28 days when stored at 5 degrees C. In addition, pH values were stable and the physical appearances of the samples did not change.

Stabilities of dobutamine, dopamine, nitroglycerin and sodium nitroprusside in disposable plastic syringes.

The solutions of dobutamine hydrochloride (5 mg/ml), dopamine hydrochloride (4 mg/ml), nitroglycerin (1 mg/ml) and sodium nitroprusside (1 mg/ml) in dextrose 5% injection were stable for 24 h when stored at 25 degrees C in 60-ml plastic syringes. For sodium nitroprusside, the syringes must be wrapped with aluminium foil (provided by the manufacturer), otherwise the loss in potency is very high (22%). There was no change in the pH values of dobutamine and dopamine solutions as well as sodium nitroprusside solutions in the prewrapped syringes. However, the pH value of nitroglycerin solutions decreased to 4.3 from 4.6 and that of sodium nitroprusside solutions in unwrapped syringes from 4.2 to 3.5; these solutions had discoloured. The chromatogram also showed new peaks from the products of decomposition. The physical appearances of the other solutions did not change.

Effect of excipients on the stability of levothyroxine sodium tablets.

Levothyroxine sodium tablets from two different manufacturers were analysed using the USP-NF method of analysis, a stability-indicating high pressure liquid chromatographic (HPLC) procedure. The results indicate that one particular manufacturer's 0.2-mg pink tablets contain some excipient(s) which act as a catalyst to hasten decomposition after extraction of levothyroxine for analysis. The same tablets from a different batch showed an additional long peak in the chromatogram, which indicated that the excipient(s) may have been changed. The same manufacturer has also used three different types of bottles/lids for the same product during the last year. Good manufacturing practice requires that new compatibilities/stability studies be conducted to assure the quality of the product. Ongoing stability studies are required by the Food and Drugs Administration (FDA). The use-life of 0.2-mg pink tablets of this manufacturer may be short.

Development of oral liquid dosage forms of acetazolamide.

Two oral liquid dosage forms of acetazolamide have been developed. Using the solubility profiles, polyethylene glycol 400 (7%, v/v) was used as the solubilizing agent and propylene glycol (53%, v/v) as the cosolvent to keep acetazolamide in solution. Because of the bitter taste of acetazolamide, sweetening agents (simple syrup, sorbitol solution, and artificial sweeteners) and flavors (raspberry, sweet, and menthol) were added to the final formulations. A buffer (either phosphate or citrate) solution was used to maintain a pH value of 4 (pH of maximum stability as reported earlier) to minimize hydrolysis. The final dosage forms were stable for at least 90 days at 37 degrees C (loss of potency of 5%). According to FDA guidelines, a tentative expiry date of 2 years at 25 degrees C is justifiable.

Chemical stability of cefotetan disodium in 5% dextrose and 0.9% sodium chloride injections.

The chemical stability of cefotetan disodium in 5% dextrose and 0.9% sodium chloride injections has been studied using a stability-indicating high-pressure liquid chromatographic (HPLC) assay method. The drug appears to be relatively unstable at 25 degrees C (expiry time 2 days), compared with at least 41 days at 5 degrees C and at least 60 days at -10 degrees C. Thawing the frozen samples in a microwave (90 s) did not cause any significant decomposition. The manufacturer's recommended expiry time of 4 days at 5 degrees C and at least 7 days at -10 degrees C is very conservative. The HPLC method developed is accurate and precise with a relative percentage standard deviation of 1.7 based on six readings. The method appears to be stability-indicating as the samples decomposed under drastic conditions had almost no drug left and new peaks were observed in the chromatograms.

Stability of acyclovir sodium in dextrose and sodium chloride injections.

The chemical stability of acyclovir sodium in dextrose 5% w/v and sodium chloride 0.9% w/v injections has been studied using a stability-indicating high-pressure liquid chromatographic (HPLC) method. The drug appears to be very stable in both admixtures. There was no decomposition after 37 days of storage at 25 degrees C or 5 degrees C. The manufacturer-recommended expiry date of 24 h at 25 degrees C is too conservative. The solutions were clear throughout the study period and the pH values had decreased slightly in both the solutions. Acyclovir appears to be very stable on the alkaline side of the pH range and less so on the acidic side. There was no loss in the potency of acyclovir when mixed with dobutamine and dopamine.