Plutonium in bone: a high resolution autoradiographic study using plutonium-241.

Plutonium-241 citrate solution at pH 6-5 was injected intravenously into hamsters and an adult rabbit at a dose of 10 kBq g-1 (260 nCi g-1). The hamsters were killed serially at 15 min, 2 hours, 1 day, 10 days, 1 month and 6 months after injection and the rabbit at 1 week. Their knee-joints or femora were examined for plutonium-241 by autoradiography. Few differences were found between the pattern of plutonium distribution in the hamsters and the rabbit. The results showed that although plutonium is initially distributed on bone surfaces, at long periods after injection it becomes deposited throughout the bone matrix. Plutonium uptake by cells in resorbing areas of periosteum, in active osteoblasts, and in chondrocytes in regions of cartilage mineralization was rapid. Plutonium concentrated more slowly on the resting bone surfaces and at sites of low metabolic activity. In addition, some unlabelled sections of skeletal tissues were immersed in a plutonium-241 citrate solution. When autoradiographed, it was found that plutonium was bound by cell nuclei, tooth enamel matrix, dentine, predentine and bone matrix. Plutonium binding to cartilage matrix was weak. The results are discussed with reference to the literature, and a model is proposed to explain the distribution pattern and fate of plutonium deposits in bone.     

The loss of transportable plutonium deposits from the macrophages of the rat femoral bone marrow

Conclusion The results showed that plutonium may be retained in bone marrow macrophages for a considerable time. Consequently, plutonium deposits in these cells may substantially irradiate components of the surrounding bone marrow and cells on bone surfaces. In red bone marrow these include the radiation sensitive cells which give rise to leukaemia. If follows that bone marrow deposits of plutonium resulting from the turnover of contaminated bone are likely to be important in radiation protection dosimetry. The period of plutonium retention in the bone marrow was found to exceed that in the liver.In addition to the above the results of this study suggest that the autoradiographic methods used to measure the plutonium content of the bone marrow are likely to be suitable for studying those factors which may affect the rate of loss of alpha-emitters from this tissue. These factors include the iron status, sex, and age of the animal and effects of drugs and radiation on the cells.     

Distribution of 239Pu in the skeleton of the tree shrew (Tupaia belangeri) between 15 and 50 months after injection

The macroscopic and microscopic distribution of intramuscularly injected, essentially monomeric, 239Pu was studied in the skeleton of the adult tree shrew (Tupaia belangeri). Data for the period between 15 and 50 months after injection are presented and compared with the data from earlier time points. Between 83 and 500 days after injection the nuclide content and the wet weight of the skeleton decreased to a constant level at about 55 per cent of the maximum values. The microscopic distribution has been analysed in distal femora, proximal humerus, proximal tibia and lumbar vertebra over the whole observation time; additionally at some selected time points proximal femur, femur shaft, distal humerus and distal tibia were analysed. The initial endosteal surface activity ranged from 3.8 to 5.3 Bq/cm2 and decreased to a minimum at about 1000 days after injection and increased thereafter. A similar behaviour was found for the dose rate near bone surfaces which was initially about 0.075 Gy/day on endosteal surfaces. In the deep bone and the deep marrow the dose rate was negligible, about 0.008 Gy/day and 0.001 Gy/day, respectively. The average cumulative dose 1500 days after injection was about 67 Gy on the endosteum, six times greater than the cumulative dose calculated from the mean concentration of plutonium in the whole skeleton. All values are normalized to an injected activity of 37 kBq/kg body weight. The tupaia data are discussed in relation to the available data from monkeys, dogs and rats.     

The relationship of bone marrow type and microvasculature to the microdistribution and local dosimetry of plutonium in the adult skeleton

Most plutonium-induced bone tumors in beagles arise in trabecular bone sites surrounded by hematopoietic tissue. The microvasculature and the relative incorporation of 241Pu (a low energy beta emitter) on trabecular bone surfaces from red and fatty marrow sites were studied in the adult beagle. The vascular volume, as determined by India ink perfusion, was about 16% in the red marrow and about 2% in the fatty marrow. The red marrow has large vascular sinusoids with a discontinuous endothelial lining whereas fatty marrow has a closed capillary bed. Plutonium concentrations on bone surfaces of red marrow sites are about 0.43 pCi/cm2/nCi/kg of injected dosage, a factor of eight greater than at fatty marrow sites (0.053 pCi/cm2/nCi/kg). The differences in the bone marrow microvasculature between red and fatty marrow help to explain differences in plutonium incorporation and local bone physiology in these skeletal sites. These observations may relate to the nonuniform distribution of plutonium-induced osteosarcomas observed in adult, long-lived mammals.     

Two-mutation models for bone cancer due to radium, strontium and plutonium

Data from beagle experiments and radium dial painters were used to derive two-mutation carcinogenesis models for bone cancer induced by the bone-seeking radionuclides radium, strontium and plutonium. For all data, the model fits indicate that at low doses both mutation rates depend linearly and equally strongly on dose rate. For the high-LET alpha-particle emitters, a cell killing term reduces the second mutation rate at high dose rates. In all cases, the combined effect of both mutation rates is a linear-quadratic dose-effect relationship for cancer at low doses. This behavior may lead to experimental data that could be mistaken as showing a threshold below which no cancers are induced. Derived parameters such as toxicity ratios and tumor growth times compare well with values reported in the literature. Furthermore, results for plutonium indicate that rapid burial of the nuclide in the growing bones of juvenile beagles leads to a significant reduction of its toxicity, as was suggested previously. The results for radium in beagles compare well with those for humans and suggest that the models derived for strontium and plutonium in beagles may be translated to humans. The significant model parameters for the accurate animal data could then also be used to fit human epidemiological data.     

Three-dimensional finite element analysis of the foot during standing--a material sensitivity study

Information on the internal stresses/strains in the human foot and the pressure distribution at the plantar support interface under loading is useful in enhancing knowledge on the biomechanics of the ankle-foot complex. While techniques for plantar pressure measurements are well established, direct measurement of the internal stresses/strains is difficult. A three-dimensional (3D) finite element model of the human foot and ankle was developed using the actual geometry of the foot skeleton and soft tissues, which were obtained from 3D reconstruction of MR images. Except the phalanges that were fused, the interaction among the metatarsals, cuneiforms, cuboid, navicular, talus, calcaneus, tibia and fibula were defined as contact surfaces, which allow relative articulating movement. The plantar fascia and 72 major ligaments were simulated using tension-only truss elements by connecting the corresponding attachment points on the bone surfaces. The bony and ligamentous structures were embedded in a volume of soft tissues. The encapsulated soft tissue was defined as hyperelastic, while the bony and ligamentous structures were assumed to be linearly elastic. The effects of soft tissue stiffening on the stress distribution of the plantar surface and bony structures during balanced standing were investigated. Increases of soft tissue stiffness from 2 and up to 5 times the normal values were used to approximate the pathologically stiffened tissue behaviour with increasing stages of diabetic neuropathy. The results showed that a five-fold increase in soft tissue stiffness led to about 35% and 33% increase in the peak plantar pressure at the forefoot and rearfoot regions, respectively. This corresponded to about 47% decrease in the total contact area between the plantar foot and the horizontal support surface. Peak bone stress was found at the third metatarsal in all calculated cases with a minimal increase of about 7% with soft tissue stiffening.     

A contact model with ingrowth control for bone remodelling around cementless stems.

This work presents a computational model for bone remodelling around cementless stems. The problem is formulated as a material optimisation problem considering the bone and stem surfaces to be in contact. To emphasise the behaviour of the bone/stem interface, the computer model detects the existence of bone ingrowth during the remodelling; consequently, the contact conditions are changed for a better interface simulation. The trabecular bone is modelled as a strictly orthotropic material with equivalent properties computed by homogenisation. The distribution of bone relative density is obtained by the minimisation of a function that considers both the bone structural stiffness and the biological cost associated with metabolic maintenance of bone tissue. The situation of multiple load conditions is considered. The remodelling law, obtained from the necessary conditions for an optimum, is derived analytically from the optimisation problem and solved numerically using a suitable finite element mesh. The formulation is applied to an implanted femur. Results of bone density and ingrowth distribution are obtained for different coating conditions. Bone ingrowth does not occur over the entire coated surfaces. Indeed, we observed regions where separation or high relative displacement occurs that preclude bone ingrowth attachment. This prediction of the model is consistent with clinical observations of bone ingrowth. Thus, this model, which detect bone ingrowth and allow modification of the interface conditions, are useful for analysis of existing stems as well as design optimisation of coating extent and location on such stems.     

Plutonium-239 distribution in the body in relation to the rhythm of administration of an iron preparation

A study was made of the dependence of plutonium distribution among organs and tissues on the time of administration of iron preparation. Iron decreased the share of plutonium in bone tissues, somewhat increased it in soft tissues, and enhanced the excretion of the radionuclide from the organism. The protective efficiency of the preparation in relation to bone tissue was associated with the time of the administration thereof.     

Comparisons of the skeletal locations of putative plutonium-induced osteosarcomas in humans with those in beagle dogs and with naturally occurring tumors in both species

Osteosarcomas occur from exposures to bone-seeking, alpha-particle-emitting isotopes, particularly plutonium. The skeletal distribution of putative 239Pu-induced osteosarcomas reported in Mayak Metallurgical and Radiochemical Plutonium Plant workers is compared with those observed in canine studies, and these are compared with distributions of naturally occurring osteosarcomas in both species. In the Mayak workers, 29% and 71% of the osteosarcomas were in the peripheral and central skeleton, respectively, with the spine having the most tumors (36%). An almost identical distribution of plutonium-induced osteosarcomas was reported for dogs injected with 239Pu as young adults. This distribution of osteosarcomas is quite different from the distributions of naturally occurring osteosarcomas for both species. In the Cooperative Osteosarcoma Study Group in humans (1,736 osteosarcomas from all ages), over 91% of the tumors occurred in the peripheral skeleton. In the Mayo Clinic group of older individuals (>40 years old), over 60% of the osteosarcomas appeared in the peripheral skeleton. The distribution of naturally occurring osteosarcomas in the canine is similar to that in the adult human. The similarities of the distributions of plutonium-associated osteosarcomas in the Mayak workers with those found in experimental studies suggest that many of the reported osteosarcomas may have been associated with plutonium exposures. These results also support the experimental paradigm that plutonium osteosarcomas have a preference for well vascularized cancellous bone sites. These sites have a greater initial deposition of plutonium, but also greater turnover due to elevated bone remodeling rates.     

Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein.

Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects.     

The uptake and redistribution of 241pu within the gonads.

Male and female hamsters and a female rabbit were injected with 241Pu citrate. The hamsters were killed serially at 15 min, 2 hours, 1 day and 10 days after injection, and the rabbit 1 week after injection. The gonads were examined for 241Pu by tissue-section autoradiography. Soon after injection the plutonium was concentrated by the contents of atretic Graafian follicles and by thecal rings in the ovary, but was found to be dispersed throughout the testes. It is suggested that the disperse distribution in the testes which is only seen soon after injection may be an artefact of tissue processing. One day after injection, plutonium was accumulated by macrophages in both the follicles of the ovary and in the interstitial tissue of the testes. Macrophages containing plutonium later migrated away from the aretic ovarian follicles towards the ovarian medulla. This pattern of distribution and redistribution in the ovary is regarded as likely to lower the effective dose from a-emitting plutonium isotopes to the viable oocytes. No migration of macrophages was seen in the testes. Histochemical staining methods revealed the presence of acid protoglycans, including chondroitin sulphate, and glycoproteins at the sites of plutonium concentration in the ovary. These molecules are regarded as likely receptor sites for plutonium. In the testes no acidic carbohydrates were found, and it is suggested that the initial binding site for plutonium may be a compound lipid. This was deduced from the apparent inability of the interstitial tissue of the testes to bind plutonium in situ.     

Possibility of a change in the parameters of plutonium-238 metabolism in the body

The influence of macro amounts of iron on 238Pu metabolism in the animal body has been studied. The data obtained indicate that 238Pu metabolism parameters change under the effect of iron. The efficiency of the agent used is demonstrated by a diminished deposition of the radionuclide in bone tissues and an increased (by 2.65 times as compared with the control) excretion of plutonium 238 in faeces.     

The applicability of the concept of morpho-physiologic metabolic factors to the modification of the distribution of 239Pu deposited in the respiratory organs

At the stage of intensive resorption of plutonium from lungs to blood the competitive per os administration of an iron preparation increased plutonium excretion from blood. As a result, soft tissues were enriched with plutonium at an early stage of metabolism. However, at the end of the experiment (30 days) the radionuclide content of soft tissues dropped to control values. Simultaneously, the share of plutonium deposited in bone tissue decreased considerably and that of the radionuclide eliminated from the body increased throughout the entire period of observation.     

Determination of the plutonium-239 content of the body

In order to increase the informativeness of the indirect dosimetric estimates of plutonium-239 body levels complex makers are widely used to enhance natural excretion of the radionuclide in urine, the ratio between 239Pu levels in urine and skeleton being measured. However, as the onset of chelate application is postponed its efficacy, with respect to the skeleton, sharply decreases making it impossible to obtain reliable information concerning plutonium 239 levels in bone tissues at later times.     

Stress relaxation behaviour of trabecular bone specimens

The present study defines several conditions under which stress relaxation tests can be performed and investigates the viscoelastic behaviour of trabecular bone in compression through a series of stress relaxation tests at three strain levels and in three loading directions of each cubic specimen. A visoelastic model is proposed to characterize the behaviour of trabecular bone and a spectrum of relaxation times is determined. Trabecular bone from the femoral head is non-linearly viscoelastic and displays anisotropic behaviour, which cannot be more symmetric elastically than orthotropic.     

Implications of the generation of reactive oxygen species by photoactivated calcein for mitochondrial studies.

Calcein is a fluorescent probe that is widely used in studies of cell viability and mitochondrial function by microscopy fluorescence imaging. It was found to have a strong photosensitizing action that prevalently involves the generation of reactive oxygen species (ROS). The photooxidation properties of calcein in solution were studied in the presence of histidine and tryptophan as oxidizable substrates. The photodegradation of histidine was mainly mediated by singlet oxygen (1O2), as shown by the inhibitory effect of sodium azide, a specific 1O2 scavenger. On the other hand, mixed photosensitization mechanisms were present when tryptophan was used as the target of the calcein-stimulated photoprocess. In addition to 1O2, hydroxyl radicals and hydrogen peroxide were involved as reactive species, as shown by using mannitol and catalase as scavengers. The calcein-photosensitized alterations of mitochondria as a potential source of artifacts in confocal microscopy studies of cells were considered. Irradiation of isolated mitochondria with visible light (500-600 nm) in the presence of calcein induced opening of the permeability transition (PT) pore. The extent of the mitochondrial membrane photodamage, however, was modulated by the nature of the calcein environment. Thus, pore opening was triggered at short irradiation times and low dye concentrations when calcein was dissolved in the bulk medium. On the contrary, calcein concentrated in the matrix space was rather inefficient as photosensitizer even at concentrations 10 times higher than those present in the external medium.     

In vitro mineralization by mesenchymal stem cells cultured on titanium scaffolds

Titanium has been utilized in the field of orthopaedic and dental reconstructive surgery, but mineralization through osteogenic differentiation of osteogenic cells on titanium surfaces has not been fully investigated. Here we cultured rat mesenchymal stem cells (MSCs) on the surfaces of titanium dishes in osteogenic media containing calcein which is a calcium-binding fluorescence dye. On titanium dishes, MSCs showed high viability to adhere to the surfaces and excellent proliferation. At day 14 of culture, MSCs differentiated into osteoblasts to form mineralized matrices on titanium dishes as well as tissue culture polystyrene (TCPS) dishes which are widely recognized as optimal culture substrates. Calcein was incorporated into the bone minerals fabricated by MSCs cultured on both substrates to show green emission under fluorescence microscopy. The fluorescence intensity was quantified with an image analyser during culture periods. These results indicate that the surfaces of titanium showed a high adhesion/proliferation potential to MSCs and that the titanium effectively supported the osteogenic differentiation of MSCs comparable to TCPS dishes. Therefore, the titanium is an effective scaffold that is applicable in bone reconstruction surgery.     

Radiation Effects on Mortality from Solid Cancers Other than Lung,Liver, and Bone Cancer in the Mayak Worker Cohort: 1948–2008

Radiation effects on mortality from solid cancers other than lung, liver, and bone cancer in the Mayak worker cohort: 1948–2008. The cohort of Mayak Production Association (PA) workers in Russia offers a unique opportunity to study the effects of prolonged low dose rate external gamma exposures and exposure to plutonium in a working age population. We examined radiation effects on the risk of mortality from solid cancers excluding sites of primary plutonium deposition (lung, liver, and bone surface) among 25,757 workers who were first employed in 1948–1982. During the period 1948–2008, there were 1,825 deaths from cancers other than lung, liver and bone. Using colon dose as a representative external dose, a linear dose response model described the data well. The excess relative risk per Gray for external gamma exposure was 0.16 (95% CI: 0.07 – 0.26) when unadjusted for plutonium exposure and 0.12 (95% CI 0.03 – 0.21) when adjusted for plutonium dose and monitoring status. There was no significant effect modification by sex or attained age. Plutonium exposure was not significantly associated with the group of cancers analyzed after adjusting for monitoring status. Site-specific risks were uncertainly estimated but positive for 13 of the 15 sites evaluated with a statistically significant estimate only for esophageal cancer. Comparison with estimates based on the acute exposures in atomic bomb survivors suggests that the excess relative risk per Gray for prolonged external exposure in Mayak workers may be lower than that for acute exposure but, given the uncertainties, the possibility of equal effects cannot be dismissed.     

Effect of plutonium-239 on the mitotic activity of mouse bone marrow cells

Summary Mitotic index of the bone marrow cells was studied in femoral bone marrow of mice given 313 kBq²³⁹Pu kg^–1. The attention was turned to the femoral midshaft and the mitose concentration, intensified by Colcemid stathmokinetic effect, was evaluated in a sampling field from endosteal surface to the central venous canal, throughout 68 weeks. It has been found that the plutonium effect in the sampling band is rather uniform except the points in subendosteal zone early after plutonium injection, where the mitotic index was reduced in such a way that the mitotic gradient, observed in controls, was affected. The mitotic activity in femoral diaphysis of plutonium injected mice was mobilized approximately till the 30th week of contamination. Later it deteriorated progressively. The results are discussed and should not be regarded as representative for the entire bone marrow hemopoiesis.     

Differences in the uptake of transferrin bound 239Pu and 59Fe into multicellular spheroids of hepatocytes from adult male rats

Hepatocytes were cultured as monolayers and multicellular spheroids, respectively. The uptake of both transferrin-bound metals, iron and plutonium, differed significantly between these two culture systems. The uptake into the multicellular spheroids for plutonium was about 30 times greater, and for iron about 4 times greater, than in monolayer-cultured hepatocytes, which is not a consequence of proliferation and/or de-differentiation of the hepatocytes in the multicellular spheroid culture system. A comparison of the iron and plutonium uptake showed that plutonium was delivered to the cells to an 8-fold greater extent than iron if the hepatocytes were cultured as spheroids. Additionally, the binding of plutonium was not inhibited by preincubation of the spheroids with the iron-transferrin complex. Therefore, we propose that there are two different binding sites for iron and plutonium on hepatocyte membranes.