Recombinant human bone morphogenetic protein-2 stimulates bone formation during interfrontal suture expansion in rabbits.

INTRODUCTION: Suture expansion stimulates bone growth to correct craniofacial deficiencies but has a high potential of treatment relapse. The objective of this study was to investigate whether there is a dose-dependent relationship between the recombinant human bone morphogenetic protein-2 (rhBMP-2) and bone formation during suture expansion. METHODS: Fifty 6-week-old male New Zealand white rabbits were randomly assigned to 5 groups to receive 0 (control), 0.01, 0.025, 0.1, or 0.4 mg/mL of rhBMP-2 delivered by absorbable collagen sponge placed over the interfrontal suture. The suture was expanded for 33 days by 200 g of constant force via a spring anchored with 2 miniscrew implants. Distance of suture expansion, suture volume, and cross-sectional area after expansion were measured using radiographs with bone markers and microcomputed tomography. Suture widths and mineralization appositional rates were calculated based on the widths between bone labels under an epifluorescent microscope. Software (Multilevel Win 2.0; University of Bristol, Bristol, United Kingdom) was used to model distance of suture expansion over time as polynomials to compare group differences. Wilcoxon signed rank tests were performed to compare the suture volume and cross-sectional area, mineral apposition rate, and suture width between groups. The significance level was set at P = 0.05. RESULTS: Whereas the sutures were expanded in all groups, sutures were expanded by significantly greater amounts in the control and the 0.01 mg/mL groups without fusing the sutures than in the 0.025, 0.1, and 0.4 mg/mL groups with fusing sutures. Compared with the controls, the 0.01 mg/mL group showed significantly lower suture volumes, cross-sectional areas, and suture widths after expansion. The mineral apposition rate was significantly higher in the 0.01 mg/mL group than in the controls from days 10 to 30. CONCLUSIONS: The 0.01 mg/mL dose of rhBMP-2 delivered by absorbable collagen sponge can stimulate bone formation at the bony edges of the suture during suture expansion; however, higher concentrations cause suture fusion. With an appropriate concentration, rhBMP-2 might facilitate suture expansion for clinical uses.

Variation of long-lived free radicals responsible for the EPR native signal in bone of aged or diseased human females and ovariectomized adult rats.

The purpose of this study was to gain insights into the variations seen in the electron paramagnetic resonance (EPR) spectroscopy of the native signals of teeth and bones used for retrospective dosimetry measurements. We determined that changes occur in the long-lived free radicals responsible for the native signal of cortical bone in aging or diseased human females and aged ovariectomized rats. This was done by measuring the magnitude of the broad (BC) and narrow (NC) components of the native EPR signal of bone following chemical extraction, aging, crushing and thermal annealing. Bone from the upper midshaft of femora of young (17-34 years old, n=5) and elderly (70-92 years old, n=18) females was examined. The results showed that the elderly women had significantly higher BC than the younger women (P<0.01). A similar interpretation was made of the data from an aging female rat osteoporosis model. The results for the NC signals were similar. Finally, dramatic decreases in both NC and BC signals were seen in HIV positive and uncontrolled diabetic (one each) patients indicating the need for studying this signal for a broad spectrum of metabolic disorders. Experiments were performed which strongly indicate that iron liganded with organic molecules is the source of the BC signal. Finally, the accuracy achieved in this study indicates that resolving the dosimetric signal (g=2.0018) should be improved by subtraction of the deconvoluted NC and BC signals from the original spectrum.

Comparison of sample preparation and signal evaluation methods for EPR analysis of tooth enamel.

In dose reconstruction by EPR dosimetry with teeth various methods are applied to prepare tooth enamel samples and to evaluate the dosimetric signal. A comparison of seven frequently used methods in EPR dosimetry with tooth enamel was performed. The participating Institutes have applied their own procedure to prepare tooth enamel samples and to evaluate the dosimetric signal. The precision of the EPR measurement and the dependence of the estimated dosimetric signal with irradiation up to 1000 mGy were compared. The obtained results are consistent among the different methods. The reproducibility of the dosimetric signal and its estimated relation with the absorbed dose was found to be very close for the applied methods with one possible exception.

Preliminary report on the development of a virtually nondestructive additive dose technique for EPR dosimetry.

We propose a new approach to the additive dose method in EPR dosimetry studies for tooth enamel specimens. We outline a specialized routine whereby the sample may be left for the most part unirradiated, while only a small aliquot of the sample will be additively irradiated to relatively large doses. The routine is done in such a way so as not to significantly compromise either precision or accuracy of the dose reconstruction. It is also demonstrated that the overall throughput of the dose reconstruction is not appreciably compromised. With this potential ability, the utility of an international dose/sensitivity standard for EPR dosimetry of teeth is considered.

Vesicle number does not predict postsynaptic measures of miniature synaptic activity frequency in cultured cortical neurons.

We tested the hypothesis that heterogeneity in the frequency of miniature synaptic activity reflects differences in the number of vesicles present in presynaptic terminals. Using imaging techniques, we measured dendritic miniature synaptic calcium transients attributed to the spontaneous release of single transmitter quanta. Following imaging, the identified neurons were processed for serial transmission electron microscopy. At sites of quantal Ca(2+) transients mediated by N-methyl-D-aspartate receptors, we confirmed the presence of excitatory synapses and measured the total number of vesicles and the number of docked vesicles. We observed no correlation between the frequency of spontaneous miniature activity and either the total vesicle number or the number of docked vesicles. We conclude that the presynaptic vesicle complement as measured by ultrastructural analysis does not necessarily determine the frequency of spontaneous activity at synapses mediated by N-methyl-D-aspartate receptors.

Ultrastructural correlates of quantal synaptic function at single CNS synapses.

We have tested the hypothesis that functional differences between synapses are associated with ultrastructure in cultured cortical neurons. Using Ca(2+) imaging, we measured NMDA receptor-mediated miniature synaptic calcium transients attributed to the spontaneous release of single transmitter quanta. After imaging, the identified neurons were processed for serial transmission electron microscopy. At sites of quantal NMDA receptor-dependent Ca(2+) transients, we confirmed the presence of excitatory synapses and measured spine size and synaptic contact area. Our results demonstrate that synapse size correlates positively with the amplitude of the NMDA receptor-mediated postsynaptic response, suggesting that larger synapses express a greater number of NMDA receptors. Therefore, regulation of quantal amplitude may involve processes that alter synapse size.

An EPR dosimetry method for rapid scanning of children following a radiation accident using deciduous teeth.

Electron paramagnetic resonance dosimetry may be applied to whole deciduous teeth of children. This makes it feasible to make direct measurement of absorbed gamma ray dose in the days and weeks following a nuclear accident, particularly if used in conjunction with a public awareness program. The technique reported here requires little sample preparation and has resulted in precision of approximately 30 mGy (1 sigma) for a deciduous incisor. Under conditions for rapid screening procedures, the methodology is estimated to provide 0.5 Gy accuracy. The largest error in the process is the determination of an appropriate background native signal for subtraction from the whole tooth spectrum. The native signal is superimposed on the radiation-induced signal, and the subtraction requires knowledge of a sample's relative content of enamel and dentin along with their relative native signal intensities. Using a composite background standard, an equivalent absorbed dose of 70+/-38 mGy (1 sigma) was determined. The lower detection limit of the technique was achieved by the elimination of anisotropic effects through rotation of the sample during measurement, together with subtraction of the standard native background signal and empty tube background spectra from the sample spectra.

EPR properties of synthetic apatites, deorganified dentine, and enamel.

Electron paramagnetic resonance spectroscopy (EPR) was used to study synthetic hydroxyapatite and approximately 1, 2, and 6% synthetic carbonated apatites, deorganified dentine, and enamel. The carbonated apatites were synthesized by hydrolysis of dicalcium phosphate. Comparisons were made with spectra from enamel and deorganified dentine. Microwave power saturation and dose responses were determined for the synthetic materials. The Marquardt version of the Levenberg decomposition method was used to extract individual signals from the apatite data. Two samples of dentine were irradiated with 25 and 100 Gy, respectively, from a 60Co source. The first sample was then deorganified at 200 degreesC using the Soxhlet extraction technique. A third sample was irradiated with 100 Gy after deorganification. The resulting EPR spectra were then compared. It was determined that the dosimetric signal of 2% synthetic carbonated apatite was approximately the same as that of enamel. It was also verified that the dosimetric signal saturates at about 2% in synthetic carbonated apatites. The study established that the precenters responsible for the dosimetric signal (g perpendicular = 2.0018, g parallel = 1.9985) are preferentially concentrated in the surface-accessible region of the mineral component, as shown by the approximately 80% attenuation of the dosimetric signal in dentine following deorganification. The precenters responsible are not destroyed by the deorganification since the magnitude of the dosimetric signal from the dentine specimen irradiated following deorganification was approximately twice that of the comparable untreated, irradiated sample. Finally, the dose response of 2 and 6% synthetic carbonated apatites was determined.

Electron paramagnetic resonance techniques and space biodosimetry.

This paper was presented at a workshop addressing the potential of biodosimetry techniques for use in the interplanetary space program. Some of the concerns for adequate dosimetry in space include: (1) a dosimeter that provides a permanent record of the cumulative dose and can be read independently on return to Earth; (2) a dosimeter which cannot be lost, forgotten or inadvertently removed by an individual; and (3) appropriate assessments of radiation exposures that pose an acute health risk and could jeopardize the success of an interplanetary mission. Tooth enamel is a permanent, stable biological dosimeter showing great promise in retrospective dosimetry of radiation accidents. With a proper technique, the minimum detectable dose can be in the range of tens of milligrays in extracted, prepared teeth. In addition to transient accidental doses, the cumulative dose from chronic low-level exposures (which individually may be below reportable limits) is recorded in the enamel of teeth. While many teeth remain with an individual over all or most of a lifetime, one or more are often removed due to dental problems and provide an opportunity to make dosimeteric measurements. The collection and analysis of extracted teeth in later life allows measurement of cumulative lifetime dose using the high-sensitivity techniques described in this paper. The goal of a lightweight, high-sensitivity, in vivo EPR spectrometer has not yet been realized, but its benefit to all aspects of retrospective dosimetry, terrestrial or otherwise, would be great. This paper reviews the current status of EPR dosimetry of teeth as applied to retrospective measurements of accidental exposures and outlines future research directions which will further reduce the limits of detection.

The first international intercomparison of EPR-dosimetry with teeth: first results.

Intercomparison of EPR-dosimetric techniques using tooth enamel had been performed in order to check whether the results produced by different laboratories are consistent and accurate. Participants were supposed to evaluate doses applied to pulverized enamel samples, using routine techniques from their laboratories. The intercomparison has demonstrated a great variety of methods used for dose reconstruction. Peculiarities of experimental approaches are discussed systematically in terms of procedure for recording the EPR-spectra, determination of the amplitude of the radiation induced signal, determination of the dose, and error propagation.

Preparation-induced errors in EPR dosimetry of enamel: pre- and post-crushing sensitivity.

Polyakov et al. (1995) showed errors in dose estimation as a function of grain size for enamel grains given beta irradiation after crushing. We tested the effect of gamma irradiation applied to the specimens before and after crushing. We confirmed Polyakov's observations and found that post-crushing irradiation altered the slope of the dose-response curve of the hydroxyapatite signal and produced a grain-size-dependent offset. No changes in the slope of the dose-response curve were seen in enamel caps irradiated whole before crushing.

Electron paramagnetic resonance dosimetry of dentine following removal of organic material.

The feasibility of using dentine from surgically extracted human teeth as in vivo dosimeters was investigated. The organic fraction of human dentine was removed by Soxhlet extraction with diethylenetriamine. The specimens were then crushed and 75 to 250 microns granules were given doses of gamma radiation ranging from 50 mGy to 8 Gy. Following irradiation, electron paramagnetic resonance spectra were collected. Signals were detected with Lande factors of g = 2.0018, line width = 0.903 mT; and g = 1.9961, line width = 0.444 mT. These signals have both been reported for hydroxyapatite of bone and enamel. Several other signals were also seen but not characterized. It was concluded that doses of 500 mGy or less may be resolved with prior removal of the organic component of dentine.

Thermoluminescence measurements of gamma-ray doses attributable to fallout from the Nevada test site using building bricks as natural dosimeters.

During the 1950's, the U.S. Government conducted an intensive atmospheric nuclear testing program in Nevada. Fallout from these atmospheric tests was measured throughout the U.S. with some of the heaviest concentrations to populated areas falling east of the test site in Washington County, UT. External exposures from 6.5 x 10(-4) C kg-1 to 26 x 10(-4) C kg-1 (2.5-5.0 R) were reported for this region. This study provides an independent measurement of fallout radiation doses to selected communities in Utah using a thermoluminescence technique originally developed for the dating of ancient pottery. The application of the predose thermoluminescence technique to fallout dosimetry is described. A mean dose of 38 +/- 15 mGy (4.4 +/- 1.7 R), attributed to fallout radiation, was measured in quartz grains extracted from the outer centimeter of bricks removed from six communities in Washington and Kane Counties in Utah.

Corpus callosum: multiple parameter measurements in rodents and humans.

A magnified drawing of a human or a rodent corpus callosum is traced on a digitizing tablet. From this tracing the computer calculates callosal area, perimeter, length, and 99 widths, one for each percentile location along the longitudinal axis of the callosum. In addition, the human program encloses the callosum within a rectangle to obtain several other measures. The use of percentile widths allows one to generate a callosum profile to compare different clinical groups or different species. The human callosum program is compared to one recently reported by another research group.

A computer-aided procedure for measuring swim rotation.

As a rat or mouse swims in a small cylinder, its movements are tracked by an observer using a joystick, and the information is sent to a Macintosh computer. The swimming circle is broken into quadrants. The sequence of quadrants entered and the time spent in each quadrant are recorded as the basic data. From the data set one can extract full or partial turns, clockwise or counterclockwise rotations, total activity, and speed of swimming clockwise and counterclockwise. Two laterality indices, one based on full turns and the other on partial turns, are calculated. Test-retest reliability for rats and mice for 3-minute and 5-minute observation intervals are reported.

A computer-aided procedure for measuring Morris maze performance.

A common procedure for recording Morris maze performance is to trace the animal's path on a template of the maze. This procedure is used in a computer-based recording system. A maze template is placed on a digitizing tablet, an electronic cursor with pen attachment is used to trace the animal's path, and the data are sent to a computer where a program called Spatial Maze obtains the following measurements: total time in seconds, total distance traveled in inches, average speed, absolute and percent time in each quadrant, absolute and percent time in each annulus, the XY coordinate information needed to reconstruct the complete path of the animal, swimming angles, and the number of rears while on the platform. These measurements are then sent to Excel for statistical and graphic analyses.

A computer-aided procedure for measuring discrimination learning.

A computer program is described for a two-choice black-white T-maze discrimination task involving 10 trials per day for 5 days. A Gellerman series of 44 semirandom L/R sequences is included within the program to specify the location of the reinforcing stimulus on each trial. A picture of the T-maze appears on the screen, and the experimenter tracks the animals's movements as it goes through the maze. At the end of the 10 trials, the following statistical information is obtained: number of initial choices into the left alley, number of correct choices, number of trials in which no choice was made, median time to make a choice, and a learning score based upon the path taken by the animal. These data are then sent to Excel for statistical processing.

Grooved titanium surfaces orient growth and migration of cells from human gingival explants.

A silicon mask-etching technique was used to prepare grooved surfaces that control the direction of outgrowths of human gingival explants. The method used to produce the grooves is excellent in terms of both the uniformity of the grooves and the control with which surfaces of the desired specifications can be obtained.