Glucose-induced inhibition of in vitro bone mineralization.

Patients with diabetes tend to have an increased incidence of osteopenia that may be related to hyperglycemia. However, little is known about how glucose may alter bone formation and osteoblast maturation. To determine whether glucose affects osteoblastic calcium deposition, MC3T3-E1 cells were incubated in media containing either a normal (5.5 mmol/L) or high glucose concentration (15 mmol/L) or mannitol (15 mmol/L), and bone nodule formation was examined. Net calcium flux was measured thrice weekly and cumulative calcium uptake was determined. Compared with control incubations, glucose significantly inhibited daily and cumulative calcium uptake into the nodules. At the time of matrix maturation, cultures undergo a rapid phase of increased calcium deposition; this was significantly inhibited by the presence of glucose. Total calcium uptake, determined by acid digestion, was also significantly inhibited by glucose. Area and number of nodules were quantitated at the end of the incubation period (day 30) by staining with Alizarin Red S calcium stain. Compared with both control and mannitol-treated cultures, the number of nodules was increased by incubation with glucose. Furthermore, both the average total nodular area and calcified nodular area of large nodules were increased by glucose. Cellular proliferation as well as the release of markers of osteoblast activity (osteocalcin and alkaline phosphatase) were determined at the end of the experimental period (day 30). Cellular proliferation and alkaline phosphatase activity was significantly increased in the presence of glucose, however, the release of osteocalcin into culture media was similar in all three groups. In conclusion, the present study shows that elevated glucose concentration present throughout the development of murine osteoblasts stimulates cellular proliferation while inhibiting calcium uptake. The result of glucose inhibition of calcium uptake suggests that bone could be structurally altered in diabetes.

Awake intubation made easy and acceptable.

This study examined the efficacy of a technique of administration of lignocaine 2% (with 1:200,000 adrenaline) to the nose, pharynx and larynx. A simple device constructed from a 22 gauge Optiva cannula attached to a medical oxygen supply via green "bubble" tubing and the barrel of a 3 ml syringe, similar to that described by Mackenzie, was used to administer aerosolized lignocaine initially via the nose and subsequently via a nasopharyngeal airway. Ten unsedated, unpremedicated volunteers were intubated. The mean time from the start of the administration of lignocaine to confirmation of placement of the endotracheal tube was 12 minutes (range 8 to 19 minutes). Intubating conditions were good and the procedure was well tolerated in all subjects. The mean dose of lignocaine was 4.8 mg.kg-1 (range 2.7 to 6.9 mg.kg-1) and plasma concentrations were well below toxic levels (highest concentration 3.12 mg.l-1). There was good haemodynamic stability and no episode of oxyhaemoglobin desaturation.

Effect of the vitamin D analogues paricalcitol and calcitriol on bone mineral in vitro.

Paricalcitol (19-nor-1,25-dihydroxyvitamin D(2)), a new vitamin D analogue, recently became available for the treatment of hyperparathyroidism in patients with end-stage renal disease. It is safe and effective in suppressing parathyroid hormone, with apparently less propensity for hypercalcemia than calcitriol (1, 25-dihydroxyvitamin D(3)). However, the mechanism of action on bone has not been fully elucidated. This study compares the effects of paricalcitol and calcitriol on the bone mineral. Neonatal (5- to 7-day-old) mouse calvariae were incubated in the absence or presence of either paricalcitol or calcitriol for 48 hours, and calcium flux, osteocalcin and acid and alkaline phosphatase activity, and interleukin-6 (IL-6) release were determined. Increasing concentrations of both calcitriol and paricalcitol increased calcium efflux. At lower concentrations, paricalcitol had no effect on acid phosphatase activity; however, at 10(-8) mol/L, paricalcitol caused a significant increase similar to that of calcitriol at 10(-9) mol/L. Increasing concentrations of paricalcitol had no effect on alkaline phosphatase activity, whereas calcitriol (10(-8) mol/L) caused significant inhibition. At low concentrations, paricalcitol had no effect on osteocalcin release; however, at 10(-8) mol/L, both compounds significantly increased osteocalcin production. Neither compound had an effect on IL-6 release. These data show that: (1) at low concentrations, both compounds induce a similar calcium efflux from cultured bone; (2) at low concentrations, paricalcitol has no effect on osteocalcin or acid and alkaline phosphatase activity; (3) at greater concentrations, paricalcitol and calcitriol have similar effects on acid phosphatase and osteocalcin activity; (4) calcitriol, but not paricalcitol, inhibits alkaline phosphatase release; and (5) the bone-resorbing effect of both compounds is independent of IL-6 release. Thus, although both compounds have similar effects on calcium efflux from bone, at therapeutic concentrations, paricalcitol does not seem to inhibit osteoblast activity. This may explain, in part, the lower calcemic effect of paricalcitol.

Role of interleukin-6 in beta2-microglobulin-induced bone mineral dissolution.

BACKGROUND: beta2-microglobulin (beta2m) amyloidosis is commonly seen in patients undergoing long-term dialysis. beta2m has been shown to induce in vitro bone mineral dissolution. The present study was designed to investigate the effect of beta2m on osteoblast function and the role of interleukin-6 (IL-6) on beta2m-induced bone resorption. METHODS: Using neonatal mouse calvariae as well as primary osteoblasts and MC 3T3 osteoblast-like cells, IL-6 production, release, and gene expression were investigated with enzyme-linked immunosorbent assay (ELISA) and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) techniques, respectively. RESULTS: In calvariae, beta2m induced a time- and dose-dependent calcium release, which was maximum following a 48-hour incubation at a concentration of 10-5 mol/L. beta2m (10-6 mol/L) also induced a significant release of IL-6 from calvarial and primary osteoblastic cultures. Using 10-6 mol/L beta2m, the amount of IL-6 mRNA in MC 3T3 cells increased in a time-dependent fashion, which peaked at 3 hours and declined to baseline by 12 hours. In primary osteoblast cells, beta2m maximally increased IL-6 mRNA levels at 6 hours; however, they remained elevated up to 24 hours. Compared with control, the presence of beta2m significantly increased cell proliferation of both primary osteoblasts and MC 3T3 cells. To investigate osteoblastic function further, osteocalcin mRNA was quantitated. Incubation with beta2m for 3 to 24 hours did not alter the amount of osteocalcin mRNA in the MC 3T3 osteoblast cells. CONCLUSION: beta2m affects bone metabolism by mechanisms that include increasing IL-6 gene expression and release, and enhancing osteoblast proliferation without affecting osteocalcin gene expression.

Cryotherapy and photocoagulation in the management of retinoblastoma: treatment failure and unusual complication.

Ophthalmologists treating children with retinoblastoma need to be aware of the limitations of new forms of therapy and their complications. For this reason the following case of a child with a small peripheral retinoblastoma that failed to respond to treatment with both cryotherapy and photocoagulation is recorded. The development of a rapidly growing juxtapapillary lesion prompted immediate enucleation. Histological examination revealed viable tumor cells remaining in the areas that were previously treated. The juxtapapillary mass consisted of optic nerve and detached peripapillary retina that were "dragged" towards the tumor by a thick fibrocellular membrane containing many myofibroblasts.