Measurement of 227Ac impurity in 225Ac using decay energy spectroscopy.

225Ac is a valuable medical radionuclide for targeted α therapy, but 227Ac is an undesirable byproduct of an accelerator-based synthesis method under investigation. Sufficient detector sensitivity is critical for quantifying the trace impurity of 227Ac, with the 227Ac/225Ac activity ratio predicted to be approximately 0.15% by end-of-bombardment (EOB). Superconducting transition edge sensor (TES) microcalorimeters offer high resolution energy spectroscopy using the normal-to-superconducting phase transition to measure small changes in temperature. By embedding 225Ac production samples in a gold foil thermally coupled to a TES microcalorimeter we can measure the decay energies of the radionuclides embedded with high resolution and 100% detection efficiency. This technique, known as decay energy spectroscopy (DES), collapses several peaks from α decays into single Q-value peaks. In practice there are more complex factors in the interpretation of data using DES, which we will discuss herein. Using this technique we measured the EOB 227Ac impurity to be (0.142 ± 0.005)% for a single production sample. This demonstration has shown that DES is a useful tool for quantitative measurements of complicated spectra.

Separation of 44Ti from proton irradiated scandium by using solid-phase extraction chromatography and design of 44Ti/44Sc generator system.

Scandium-44g (half-life 3.97h [1]) shows promise for positron emission tomography (PET) imaging of longer biological processes than that of the current gold standard, 18F, due to its favorable decay parameters. One source of 44gSc is the long-lived parent nuclide 44Ti (half-life 60.0 a). A 44Ti/44gSc generator would have the ability to provide radionuclidically pure 44gSc on a daily basis. The production of 44Ti via the 45Sc(p,2n) reaction requires high proton beam currents and long irradiation times. Recovery and purification of no-carrier added (nca) 44Ti from scandium metal targets involves complex separation chemistry. In this study, separation systems based on solid phase extraction chromatography were investigated, including branched diglycolamide (BDGA) resin and hydroxamate based ZR resin. Results indicate that ZR resin in HCl media represents an effective 44Ti/44gSc separation system.

Large scale accelerator production of 225Ac: Effective cross sections for 78-192MeV protons incident on 232Th targets.

Actinium-225 and 213Bi have been used successfully in targeted alpha therapy (TAT) in preclinical and clinical research. This paper is a continuation of research activities aiming to expand the availability of 225Ac. The high-energy proton spallation reaction on natural thorium metal targets has been utilized to produce millicurie quantities of 225Ac. The results of sixteen irradiation experiments of thorium metal at beam energies between 78 and 192MeV are summarized in this work. Irradiations have been conducted at Brookhaven National Laboratory (BNL) and Los Alamos National Laboratory (LANL), while target dissolution and processing was carried out at Oak Ridge National Laboratory (ORNL). Excitation functions for actinium and thorium isotopes, as well as for some of the fission products, are presented. The cross sections for production of 225Ac range from 3.6 to 16.7mb in the incident proton energy range of 78-192MeV. Based on these data, production of curie quantities of 225Ac is possible by irradiating a 5.0gcm-2 232Th target for 10 days in either BNL or LANL proton irradiation facilities.

In vivo quantification of gentamicin released from an implant coating.

Drug-releasing implants are gaining increasing interest. The present study reports a detailed physicochemical analysis of a polymeric coating based on poly(D,L-lactide) and the incorporated gentamicin combined with an in vitro and in vivo study of the gentamicin release. Differential scanning calorimeter, Fourier transform infrared spectroscopy, gel permeation chromatography and high-performance liquid chromatography showed no effect of the gamma sterilisation on the coating components or an interaction of the polymer and the gentamicin. Microbiological analysis revealed an inhibition of bacterial growth on the implant surface. For the in vivo study, gentamicin-coated wires were implanted into the tibiae of rats and harvested at different time points up to day 42. To monitor the release in vivo, gentamicin was quantified in serum, bone, endosteum, kidney, and on the explanted wires. Gentamicin was detectable over a time period of 42 days in the endosteum, up to seven days in the kidney, up to 4 h in the bone and at the end of the experiment on one of eight wires. The locally released gentamicin caused no histological changes of the kidney. Microbiologically active concentrations of released gentamicin were found in the endosteum up to 4 h after implantation. The combination of different methods supports the individual results, where quantification is complemented by visualisation or antimicrobial activity. This work demonstrates that the coating procedure results in no substantial alteration of the incorporated drug and that the in vitro burst release occurs also in vivo.

Application of ion exchange and extraction chromatography to the separation of actinium from proton-irradiated thorium metal for analytical purposes.

Actinium-225 (t1/2=9.92d) is an α-emitting radionuclide with nuclear properties well-suited for use in targeted alpha therapy (TAT), a powerful treatment method for malignant tumors. Actinium-225 can also be utilized as a generator for (213)Bi (t1/2 45.6 min), which is another valuable candidate for TAT. Actinium-225 can be produced via proton irradiation of thorium metal; however, long-lived (227)Ac (t1/2=21.8a, 99% ß(-), 1% α) is co-produced during this process and will impact the quality of the final product. Thus, accurate assays are needed to determine the (225)Ac/(227)Ac ratio, which is dependent on beam energy, irradiation time and target design. Accurate actinium assays, in turn, require efficient separation of actinium isotopes from both the Th matrix and highly radioactive activation by-products, especially radiolanthanides formed from proton-induced fission. In this study, we introduce a novel, selective chromatographic technique for the recovery and purification of actinium isotopes from irradiated Th matrices. A two-step sequence of cation exchange and extraction chromatography was implemented. Radiolanthanides were quantitatively removed from Ac, and no non-Ac radionuclidic impurities were detected in the final Ac fraction. An (225)Ac spike added prior to separation was recovered at ≥ 98%, and Ac decontamination from Th was found to be ≥ 10(6). The purified actinium fraction allowed for highly accurate (227)Ac determination at analytical scales, i.e., at (227)Ac activities of 1-100 kBq (27 nCi to 2.7 µCi).

Local gentamicin application does not interfere with bone healing in a rat model.

For the prophylaxis and treatment of bony infections antibiotics are locally used. Since several decades antibiotics mixed with bone cement (methylmethacrylate) are successfully used in prosthetic surgery and a gentamicin coated tibial nail is approved in Europe for fracture stabilization. The goal of the present study was to investigate if gentamicin, locally applied from a polymeric coating of intramedullary nails, might interfere with the bone healing process. Female Sprague Dawley rats (n = 72) were used and the tibiae were intramedullary stabilized with Kirschner-wires (k-wires) after osteotomy. This model was established earlier and shows a delayed healing with a prolonged inflammatory reaction. The open approach is clinically more relevant compared to a closed one because it mimics the clinically critical case of an open fracture, which has a higher risk of infection. The k-wire was either coated with the polymer poly(d,l-lactide) (control group) or with 10% gentamicin incorporated into the polymer (gentamicin group). In vivo µCT analyses were performed at days 10, 28, 42, and 84 after osteotomy. Mechanical torsional testing and histological evaluation were done at the days of sacrifice: 28, 42, and 84. The µCT analyses revealed an increase in tissue mineral density (TMD) over the healing period in both groups. In the control group, the torsional stiffness and maximum load did not reach the values of the intact contralateral side at any time point. At day 84 the gentamicin treated tibiae, however, showed significantly better maximum load compared to the control group. The histology showed no bony bridging in the control, whereas in 2 of 5 calluses of the gentamicin group mineralized bridging occurred. Significantly more mineralized tissue was measured in the gentamicin group. This study shows that the local gentamicin application does not negatively interfere with the long term healing process. Local infection prophylaxis is effective without negative effects on bone healing.

Proton-induced cross sections relevant to production of 225Ac and 223Ra in natural thorium targets below 200 MeV.

Cross sections for (223,)(225)Ra, (225)Ac and (227)Th production by the proton bombardment of natural thorium targets were measured at proton energies below 200 MeV. Our measurements are in good agreement with previously published data and offer a complete excitation function for (223,)(225)Ra in the energy range above 90 MeV. Comparison of theoretical predictions with the experimental data shows reasonable-to-good agreement. Results indicate that accelerator-based production of (225)Ac and (223)Ra below 200 MeV is a viable production method.

225Ac and 223Ra production via 800 MeV proton irradiation of natural thorium targets.

Cross sections for the formation of (225,227)Ac, (223,225)Ra, and (227)Th via the proton bombardment of natural thorium targets were measured at a nominal proton energy of 800 MeV. No earlier experimental cross section data for the production of (223,225)Ra, (227)Ac and (227)Th by this method were found in the literature. A comparison of theoretical predictions with the experimental data shows agreement within a factor of two. Results indicate that accelerator-based production of (225)Ac and (223)Ra is a viable production method.

Radioarsenic from a portable (72)Se/(72)As generator: a current perspective.

Positron emission tomography (PET) of slower biological processes calls for the use of longer lived positron emitting radioisotopes. Beyond radionuclide production considerations, practicality and rapidity of subsequent labeling chemistry further limits the selection of radioisotopes with potentially favorable nuclear properties. One additional limitation is the availability of PET radiotracers at the point-of-care with appropriate on-site production methodologies or robust radionuclide generator systems. The positron emitter (72)As (half-life 26 h) is generated via decay of (72)Se (half-life 8.5 d); this pair comprises and excellent generator system for clinical availability of a longer lived PET isotope. Many (72)Se/As generator systems have been introduced utilizing the rich interplay of Se(IV)/Se(VI) and As(III) /As(V) chemical behavior. This paper describes available generator concepts, and briefly outlines some current arsenic labeling methodologies for the introduction of radioarsenic into biomolecules.

Selenium-72 formation via nat Br(p,x) induced by 100 MeV protons: steps towards a novel 72Se/72As generator system.

Selenium-72 production by the proton bombardment of a natural NaBr target has been successfully demonstrated at the Los Alamos National Laboratory Isotope Production Facility (LANL-IPF). Arsenic-72 (half life 26 h) is a medium-lived positron emitting radionuclide with the major advantage of being formed as the daughter of another "generator" radioisotope (Se-72, 8.5 d). A (72)Se/(72)As generator would be the preferred mechanism for clinical utilization of (72)As for positron emission tomography (PET). No portable (72)Se/(72)As generator system has been demonstrated for convenient, repeated (72)As elution ("milking"). In this work, we describe (72)Se production and recovery from irradiated NaBr targets using a 100 MeV proton beam. We also introduce an (72)As generator principle based on (72)Se chelation followed by liquid-liquid extraction, which will be transferred to a solid-phase sorption/elution system.

Proton beam simulation with MCNPX/CINDER'90: Germanium metal activation estimates below 30MeV relevant to the bulk production of arsenic radioisotopes.

Germanium metal targets encapsulated in Nb shells were irradiated in a proton beam. Proton and secondary neutron beam fluences as well as radionuclide activity formation were modeled using MCNPX in combination with CINDER90. Targets were chemically processed using distillation and anion exchange. Good agreement between the measured radiochemical yields and MCNPX/CINDER90 estimates was observed. A target of pentavalent (73,74)As radioarsenic for neutron activation studies was prepared.

Local inhibition of angiogenesis results in an atrophic non-union in a rat osteotomy model.

Long bone and in particular tibia fractures frequently fail to heal. A disturbed revascularisation is supposed to be a major cause for impaired bone healing or the development of non-unions. We aim to establish an animal model, which reliably mimics the clinical situation. Human microvascular endothelial cells (HMEC-1) and primary human osteoblast like cells (POBs) were cultured with different angiogenesis-inhibitors (Fumagillin, SU5416, Artesunate and 3,5,4'-Trimethoxystilbene) released out of poly(D,L-Lactide) (PDLLA) coated k-wires and cell activity was determined. Discs containing PDLLA or PDLLA + Fumagillin/Artesunate were placed at the chorionallantoic membrane of hen eggs and the effect on vessel formation and egg vitality was observed. Tibia osteotomy was performed in rats and stabilised with K-wires coated with PDLLA + Fumagillin or with PDLLA only (control group). The healing was compared at different time points to the PDLLA control. Fumagillin and Artesunate inhibited the activity of HMEC-1 with minor effect on POBs. Artesunate caused embryonic death, whereas Fumagillin had no effects on egg vitality, but reduced the blood vessels. In the animal study all rats showed an impaired healing with reduced biomechanical stability. The Fumagillin treated tibiae had a significantly decreased callus size at day 42 and 84, less blood vessels in the early callus, a reduced histological callus size at day 10, 28 and 84, as well as an altered callus composition. This study presents a less vascularised, atrophic, tibia non-union and can be used in further investigations to analyse the pathology of atrophic non-union and to test new interventions.

Local BMP-2 application can rescue the delayed osteotomy healing in a rat model.

Delayed healing is still a severe complication in the clinic. The aim of the present study was to investigate the effect of locally delivered BMP-2 incorporated in a poly(d,l-lactide) (PDLLA) implant coating in a rat model with delayed tibial healing. The healing delay in this model is not caused by mechanical instability or additional tissue manipulation and presents therefore a common and challenging clinical situation of impaired healing. Radiological, histological and biomechanical evaluations were performed at days 5, 10, 28, 42, and 84 after tibial osteotomy. The control group showed a delayed healing without complete bridging and without reaching the biomechanical stability of the contralateral tibiae after 84 days. The mechanical stability of the BMP-treated tibiae showed a significant increase at days 28 and 42 compared to the control group and exceeded the stability of the intact contralateral tibiae. Less cartilage was detected at day 28 and the mineralisation was significantly enhanced at day 42 due to the local BMP application. Looking at the early healing phase (day 10) a reduced vascularisation was seen in the BMP group. This reflects the situation seen during normal healing, whereas the delayed healing in the present model had an increased vascularisation. The present study clearly demonstrates that local BMP-2 application can stimulate delayed healing in a clinically relevant animal model.

Cryopreservation of ovarian tissue: an emerging technology for female germline preservation of endangered species and breeds.

Many hundreds of exotic species and domestic animal breeds have been lost over the course of the last few decades. In order to avoid a similar fate to other animals threatened with extinction, it is crucial to develop and apply rescue strategies to ensure their survival for the future. One option as a safeguard measure is the cryopreservation of the main source of female gametes enclosed within the ovary: the primordial follicles. So far, there are three options to cryopreserve small ovarian follicles: whole ovary, ovarian cortical tissue or isolated follicles, with the use of slow freezing or vitrification methods. After cryopreservation, the harvested material can be transplanted or cultured, with the aim to produce mature fertilizable oocytes. The objective of this review is to summarize the current status of the cryopreservation of ovarian tissue in domestic species and non-endangered wild mammals as model for threatened and endangered species and breeds, and to provide new insights into techniques that can be applied in the future.

Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males.

This study aimed to establish artificial insemination (AI) protocols to predictably initiate pregnancy during the breeding season in the European brown hare (EBH) (Lepus europaeus PALLAS, 1778). Semen was collected from seven captive and eight free-ranging males by means of electroejaculation. Semen from the free-ranging males was cryopreserved using directional freezing. Total motility/integrity of fresh and frozen-thawed semen was 91.6%/87.7% and 46.9%/53.8%, respectively. Ovulation was induced in ultrasonographically preselected females using a gonadotropin-releasing hormone analogue. Each female was inseminated with 1 mL fresh (Group A, n=16) or frozen-thawed semen (Group B, n=9) at a concentration of 100x10(6) spermatozoa/mL. The use of ultrasonography (10 to 22 MHz) confirmed the intracervical semen deposit, the success of artificial ovulation induction (formation of postovulatory corpus luteum), and permitted the monitoring of individual pregnancies. Although sperm motility/integrity was significantly different between groups, no significant difference was detected in conception rates (A, 87.50%; B, 77.78%). Because of embryonic resorption, there was a slight difference in fertility rate between groups (A, 62.5%; B, 77.78%). Overall, AI in captive EBH using fresh and frozen-thawed semen achieved successful fertility rates. Long-term cryopreserved semen was used to bring new genetic material from the wild into a genetically limited captive population without extensive animal transport. Therefore, AI has the potential to enhance breeding programs for EBH especially when cryopreserved semen from wild donors is used.

[Minimally invasive T-plating at the proximal humerus]. / Minimalinvasive T-Plattenosteosynthese am proximalen Humerus.

BACKGROUND: A generally accepted recommendation for the optimum surgical treatment of unstable, dislocated fractures of the proximal humerus is not yet available. According to the general surgical trend to minimally invasive techniques, the object of this study was to conduct follow-up examinations of the technique of minimally invasive T-plating at the proximal humerus developed at our hospital and to compare the results with those of other osteosynthetic techniques, especially concerning the complications of the surgical approach and the clinical outcome. PATIENTS AND METHOD: Between 1/2000 and 6/2003, 117 minimally invasive T-plating procedures were performed on 116 patients by sliding through a T-plate from a small proximal incision. After application of the exclusion criteria, a total of 100 operations on 99 patients were left. The follow-up examinations were based on the Constant score and the DASH score. RESULTS: At an average value of 70 points (SD +/- 21), the results of the Constant-Murley score were only medium to satisfactory (min. 17/max. 100 points). At an average value of 32 points (SD +/- 23), the results of the DASH score also were only adequate (min. 0/max. 78 points). A correlation between age, sex or fracture classification and the score results could not be deduced. CONCLUSION: The expectations for this technique were not met. Minimally invasive T-plating could not be shown to be superior to other techniques.

[Prevalence of anti-Yersinia antibodies in European brown hares in North-Rhine Westphalia, Germany]. / Prävalenz von Anti-Yersinia-Antikörpern bei Feldhasen in Nordrhein-Westfalen.

Yersinia (Y.) pseudotuberculosis infections may lead to significant lethality in European brown hare (Lepus europaeus, Pallas) populations especially during the cold and wet seasons. In recent decades, also Y. enterocolitica was isolated from hares found dead. Consequently, a Western-blot technique proved to be valuable for the detection of antibodies against all pathogenic Yersinia isolates was applied to monitor the prevalence of antibodies in hare populations in North-Rhine Westphalia, Germany. A total of 89.6% of the 230 animals tested was seropositive. Further investigations should be performed to elucidate the role of subclinical yersiniosis in the decline of European brown hare populations in Germany.

Seasonal timing of sperm production in roe deer: interrelationship among changes in ejaculate parameters, morphology and function of testis and accessory glands.

Roe deer are seasonal breeders with a short rutting season from mid-July to mid-August. The seasonality of reproductive activity in males is associated with cyclic changes between growth and involution of both testes and the accessory sex glands. This study characterizes morphological and functional parameters of these organs prior to, during and after breeding season in live adult roe deer bucks. Size and morphology of the reproductive tract was monitored monthly by transcutaneous (testes, epididymis) and transrectal (accessory glands) ultrasonography. Semen was collected by electroejaculation. Concentration, motility and morphological integrity of spermatozoa as well as the content of proteins and testosterone in semen plasma were evaluated. Proportions of haploid, diploid and tetraploid cells were estimated by flow cytometry in testicular tissue biopsies. Serum testosterone was measured by enzyme immunoassay. Most parts of the male reproductive tract showed distinct circannual changes in size and texture. These changes were most pronounced in the testes, seminal vesicles, and prostate. All reproductive organs were highly developed during the rut only. The volume of ejaculates, total sperm number and percentages of motile and intact spermatozoa also showed a maximum during this period and corresponded with high proportions of haploid cells in the testis. The highest percentages of tetraploid cells were found in the prerutting period. The production of motile and intact spermatozoa correlated with both the protein content of semen plasma and the concentration of testosterone in semen plasma and blood serum. These results suggest the importance of combined actions of the testes and accessory sex glands and the crucial role of testosterone in facilitating the optimal timing of intensified semen production to ensure sufficient numbers of normal spermatozoa in seasonal breeders.

Different behaviour of 63Ni and 59Fe during absorption in iron-deficient and iron-adequate jejunal rat segments ex vivo.

Nickel exhibits low oral toxicity. It shares the absorptive pathways for iron, though there are substantial quantitative differences in handling of both metals. To analyse these differences more closely, jejunal segments from iron-deficient and iron-adequate rats were luminally perfused ex vivo with 59Fe and 63Ni at six different concentrations (1-500 micromo1/l) under steady state conditions. 59Fe over-all absorption increased 2.0-4.6-fold in iron-deficiency at luminal concentrations between 1 and 100 micromol/l, while 63Ni absorption increased to a much lower extent (2.6-fold at 1 micromol/l and 1.5-fold at higher luminal concentrations). Moreover, there was a 5-7-fold higher concentration for 63Ni in the jejunal tissue than in the absorbate at luminal concentrations above 50 micromol/l which was not observed at 1 micromol 63Ni/l and not for 59Fe. 63Ni tissue load showed a linear and a saturable fraction. In iron-deficiency the saturable 63Ni fraction increased 4-fold as compared to only 1.5-fold increments for 59Fe. Moreover, a substantially higher share of 63Ni was retained in the jejunal tissue at high as compare to low luminal concentrations after perfusion had been continued without luminal radioactivity. This was not found for 59Fe and suggests a concentration-dependent block of 63Ni export across the enterocytes' basolateral membrane. To explain these results one may speculate that 63Ni may bind more tightly to tissue ligands than 59Fe due to the higher thermodynamic and kinetic stability of nickel complexes. In particular, nickel may bind to a basolateral population of metal carriers and block its own basolateral transfer in a concentration-dependent manner. Tight 63Ni binding to non-specific jejunal ligands is responsible for the unaltered high linear fraction of jejunal 63Ni load in iron-deficient and iron-adequate segments. Binding of 63Ni to food and tissue ligands in the small intestine may, thus, be a likely explanation for the low oral nickel toxicity.