Combining BNCT with carbonic anhydrase inhibition for mesothelioma treatment: Synthesis, in vitro, in vivo studies of ureidosulfamido carboranes.

Mesothelioma is a malignant neoplasm of mesothelial cells caused by exposure to asbestos. The average survival time after diagnosis is usually nine/twelve months. A multi-therapeutic approach is therefore required to treat and prevent recurrence. Boronated derivatives containing a carborane cage, a sulfamido group and an ureido functionality (CA-USF) have been designed, synthesised and tested, in order to couple Boron Neutron Capture Therapy (BNCT) and the inhibition of Carbonic Anhydrases (CAs), which are overexpressed in many tumours. In vitro studies showed greater inhibition than the reference drug acetazolamide (AZ). To increase solubility in aqueous media, CA-USFs were used as inclusion complexes of hydroxypropyl ß-cyclodextrin (HP-ß-CD) in all the inhibition and cell experiments. BNCT experiments carried out on AB22 (murine mesothelioma) cell lines showed a marked inhibition of cell proliferation by CA-USFs, and in one case a complete inhibition of proliferation twenty days after neutron irradiation. Finally, in vivo neutron irradiation experiments on a mouse model of mesothelioma demonstrated the efficiency of combining CA IX inhibition and BNCT treatment. Indeed, a greater reduction in tumour mass was observed in treated mice compared to untreated mice, with a significant higher effect when combined with BNCT. For in vivo experiments CA-USFs were administered as inclusion complexes of higher molecular weight ß-CD polymers thus increasing the selective extravasation into tumour tissue and reducing clearance. In this way, boron uptake was maximised and CA-USFs demonstrated to be in vivo well tolerated at a therapeutic dose. The therapeutic strategy herein described could be expanded to other cancers with increased CA IX activity, such as melanoma, glioma, and breast cancer.

A compact scintillator-based detector with collimator and shielding for dose monitoring in boron neutron capture therapy.

Boron neutron capture therapy exploits 10B(n,α)7Li reactions for targeted tumor destruction. In this work, we aimed at developing a dose monitoring system based on the detection of 478 keV gamma rays emitted by the reactions, which is very challenging due to the severe background present. We investigated a compact gamma-ray detector with a pinhole collimator and shielding housing. Experimental nuclear reactor measurements involved varying boron concentrations and artificial shifts of the sources. The system successfully resolved the 478 keV photopeak and detected 1 cm lateral displacements, confirming its suitability for precise boron dose monitoring.

Combined BNCT-CIRT treatment planning for glioblastoma using the effect-based optimization.

Objective. Boron neutron capture therapy (BNCT) and carbon ion radiotherapy (CIRT) are emerging treatment modalities for glioblastoma. In this study, we investigated the methodology and feasibility to combine BNCT and CIRT treatments. The combined treatment plan illustrated how the synergistic utilization of BNCT's biological targeting and CIRT's intensity modulation capabilities could lead to optimized treatment outcomes.Approach. The Monte Carlo toolkit, TOPAS, was employed to calculate the dose distribution for BNCT, while matRad was utilized for the optimization of CIRT. The biological effect-based approach, instead of the dose-based approach, was adopted to develop the combined BNCT-CIRT treatment plans for six patients diagnosed with glioblastoma, considering the different radiosensitivity and fraction. Five optional combined treatment plans with specific BNCT effect proportions for each patient were evaluated to identify the optimal treatment that minimizes damage on normal tissue.Main results. Individual BNCT exhibits a significant effect gradient along with the beam direction in the large tumor, while combined BNCT-CIRT treatments can achieve uniform effect delivery within the clinical target volume (CTV) through the effect filling with reversed gradient by the CIRT part. In addition, the increasing BNCT effect proportion in combined treatments can reduce damage in the normal brain tissue near the CTV. Besides, the combined treatments effectively minimize damage to the skin compared to individual BNCT treatments.Significance. The initial endeavor to combine BNCT and CIRT treatment plans is achieved by the effect-based optimization. The observed advantages of the combined treatment suggest its potential applicability for tumors characterized by pleomorphic, infiltrative, radioresistant and voluminous features.

Study of Alternative Imaging Methods for In Vivo Boron Neutron Capture Therapy.

Boron Neutron Capture Therapy (BNCT) is an innovative and highly selective treatment against cancer. Nowadays, in vivo boron dosimetry is an important method to carry out such therapy in clinical environments. In this work, different imaging methods were tested for dosimetry and tumor monitoring in BNCT based on a Compton camera detector. A dedicated dataset was generated through Monte Carlo tools to study the imaging capabilities. We first applied the Maximum Likelihood Expectation Maximization (MLEM) iterative method to study dosimetry tomography. As well, two methods based on morphological filtering and deep learning techniques with Convolutional Neural Networks (CNN), respectively, were studied for tumor monitoring. Furthermore, clinical aspects such as the dependence on the boron concentration ratio in image reconstruction and the stretching effect along the detector position axis were analyzed. A simulated spherical gamma source was studied in several conditions (different detector distances and boron concentration ratios) using MLEM. This approach proved the possibility of monitoring the boron dose. Tumor monitoring using the CNN method shows promising results that could be enhanced by increasing the training dataset.

Calculation of the DNA damage yield and relative biological effectiveness in boron neutron capture therapy via the Monte Carlo track structure simulation.

Objective.Boron neutron capture therapy (BNCT) is an advanced cellular-level hadron therapy that has exhibited remarkable therapeutic efficacy in the treatment of locally invasive malignancies. Despite its clinical success, the intricate nature of relative biological effectiveness (RBE) and mechanisms responsible for DNA damage remains elusive. This work aims to quantify the RBE of compound particles (i.e. alpha and lithium) in BNCT based on the calculation of DNA damage yields via the Monte Carlo track structure (MCTS) simulation.Approach. The TOPAS-nBio toolkit was employed to conduct MCTS simulations. The calculations encompassed four steps: determination of the angle and energy spectra on the nuclear membrane, quantification of the database containing DNA damage yields for ions with specific angle and energy, accumulation of the database and spectra to obtain the DNA damage yields of compound particles, and calculation of the RBE by comparison yields of double-strand break (DSB) with the reference gamma-ray. Furthermore, the impact of cell size and microscopic boron distribution was thoroughly discussed.Main results. The DSB yields induced by compound particles in three types of spherical cells (radius equal to 10, 8, and 6µm) were found to be 13.28, 17.34, 22.15 Gy Gbp-1for boronophenylalanine (BPA), and 1.07, 3.45, 8.32 Gy Gbp-1for sodium borocaptate (BSH). The corresponding DSB-based RBE values were determined to be 1.90, 2.48, 3.16 for BPA and 0.15, 0.49, 1.19 for BSH. The calculated DSB-based RBE showed agreement with experimentally values of compound biological effectiveness for melanoma and gliosarcoma. Besides, the DNA damage yield and DSB-based RBE value exhibited an increasing trend as the cell radius decreased. The impact of the boron concentration ratio on RBE diminished once the drug enrichment surpasses a certain threshold.Significance. This work is potential to provide valuable guidance for accurate biological-weighted dose evaluation in BNCT.

Microdosimetric Analysis for Boron Neutron Capture Therapy via Monte Carlo Track Structure Simulation with Modified Lithium Cross-sections.

Boron neutron capture therapy (BNCT) is a cellular-level hadron therapy achieving therapeutic effects via the synergistic action of multiple particles, including Lithium, alpha, proton, and photon. However, evaluating the relative biological effectiveness (RBE) in BNCT remains challenging. In this research, we performed a microdosimetric calculation for BNCT using the Monte Carlo track structure (MCTS) simulation toolkit, TOPAS-nBio. This paper reports the first attempt to derive the ionization cross-sections of low-energy (>0.025 MeV/u) Lithium for MCTS simulation based on the effective charge cross-section scalation method and phenomenological double-parameter modification. The fitting parameters λ1=1.101,λ2=3.486 were determined to reproduce the range and stopping power data from the ICRU report 73. Besides, the lineal energy spectra of charged particles in BNCT were calculated, and the influence of sensitive volume (SV) size was discussed. Condensed history simulation obtained similar results with MCTS when using Micron-SV while overestimating the lineal energy when using Nano-SV. Furthermore, we found that the microscopic boron distribution can significantly affect the lineal energy for Lithium, while the effect for alpha is minimal. Similar results to the published data by PHITS simulation were observed for the compound particles and monoenergetic protons when using micron-SV. Spectra with nano-SV reflected that the different track densities and absorbed doses in the nucleus together result in the dramatic difference in the macroscopic biological response of BPA and BSH. This work and the developed methodology could impact the research fields in BNCT where understanding radiation effects is crucial, such as the treatment planning system, source evaluation, and new boron drug development.

A novel pH sensitive theranostic PLGA nanoparticle for boron neutron capture therapy in mesothelioma treatment.

This study aims to develop poly lactic-co-glycolic acid (PLGA) nanoparticles with an innovative imaging-guided approach based on Boron Neutron Capture Therapy for the treatment of mesothelioma. The herein-reported results demonstrate that PLGA nanoparticles incorporating oligo-histidine chains and the dual Gd/B theranostic agent AT101 can successfully be exploited to deliver a therapeutic dose of boron to mesothelioma cells, significantly higher than in healthy mesothelial cells as assessed by ICP-MS and MRI. The selective release is pH responsive taking advantage of the slightly acidic pH of the tumour extracellular environment and triggered by the protonation of imidazole groups of histidine. After irradiation with thermal neutrons, tumoral and healthy cells survival and clonogenic ability were evaluated. Obtained results appear very promising, providing patients affected by this rare disease with an improved therapeutic option, exploiting PLGA nanoparticles.

Potentialities of High-Resolution 3-D CZT Drift Strip Detectors for Prompt Gamma-Ray Measurements in BNCT.

Recently, new high-resolution cadmium-zinc-telluride (CZT) drift strip detectors for room temperature gamma-ray spectroscopic imaging were developed by our group. The CZT detectors equipped with orthogonal anode/cathode collecting strips, drift strips and dedicated pulse processing allow a detection area of 6 × 20 mm2 and excellent room temperature spectroscopic performance (0.82% FWHM at 661.7 keV). In this work, we investigated the potentialities of these detectors for prompt gamma-ray spectroscopy (PGS) in boron neutron capture therapy (BNCT). The detectors, exploiting the measurement of the 478 keV prompt gamma rays emitted by 94% 7Li nuclides from the 10B(n, α)7Li reaction, are very appealing for the development of single-photon emission computed tomography (SPECT) systems and Compton cameras in BNCT. High-resolution gamma-ray spectra from 10B samples under thermal neutrons were measured at the T.R.I.G.A. Mark II research nuclear reactor of the University of Pavia (Italy).

Multimodal evaluation of 19F-BPA internalization in pancreatic cancer cells for boron capture and proton therapy potential applications.

PURPOSE: One of the obstacles to the application of Boron Neutron Capture Therapy (BNCT) and Proton Boron Fusion Therapy (PBFT) concerns the measurement of borated carriers' biodistribution. The objective of the present study was to evaluate the in vitro internalization of the 19F-labelled p-boronophenylalanine (19F-BPA) in the human cancer pancreatic cell line (PANC-1) for the potential application of BNCT and PBFT in pancreatic cancer. The 19F-BPA carrier has the advantage that its bio-distribution may be monitored in vivo using 19F-Nuclear Magnetic Resonance (19F NMR). MATERIALS AND METHODS: The 19F-BPA internalization in PANC-1 cells was evaluated using three independent techniques on cellular samples left in contact with growing medium enriched with 13.6 mM 19F-BPA corresponding to a 11B concentration of 120 ppm: neutron autoradiography, which quantifies boron; liquid chromatography hyphenated to tandem mass spectrometry and UV-Diode Array Detection (UV-DAD), which quantifies 19F-BPA molecule; and 19F NMR spectroscopy, which detects fluorine nuclei. RESULTS: Our studies suggested that 19F-BPA is internalized by PANC-1 cells. The three methods provided consistent results of about 50% internalization fraction at 120 ppm of 11B. Small variations (less than 15%) in internalization fraction are mainly dependent on the proliferation state of the cells. CONCLUSIONS: The ability of 19F NMR spectroscopy to study 19F-BPA internalization was validated by well-established independent techniques. The multimodal approach we used suggests 19F-BPA as a promising BNCT/PBFT carrier for the treatment of pancreatic cancer. Since the quantification is performed at doses useful for BNCT/PBFT, 19F NMR can be envisaged to monitor 19F-BPA bio-distribution during the therapy.

In Vivo Evaluation of Multifunctional Gold Nanorods for Boron Neutron Capture and Photothermal Therapies.

The incidence and mortality of cancer demand more innovative approaches and combination therapies to increase treatment efficacy and decrease off-target side effects. We describe a boron-rich nanoparticle composite with potential applications in both boron neutron capture therapy (BNCT) and photothermal therapy (PTT). Our strategy is based on gold nanorods (AuNRs) stabilized with polyethylene glycol and functionalized with the water-soluble complex cobalt bis(dicarbollide) ([3,3'-Co(1,2-C2B9H11)2]-), commonly known as COSAN. Radiolabeling with the positron emitter copper-64 (64Cu) enabled in vivo tracking using positron emission tomography imaging. 64Cu-labeled multifunctionalized AuNRs proved to be radiochemically stable and capable of being accumulated in the tumor after intravenous administration in a mouse xenograft model of gastrointestinal cancer. The resulting multifunctional AuNRs showed high biocompatibility and the capacity to induce local heating under external stimulation and trigger cell death in heterogeneous cancer spheroids as well as the capacity to decrease cell viability under neutron irradiation in cancer cells. These results position our nanoconjugates as suitable candidates for combined BNCT/PTT therapies.

A Novel Approach to Design and Evaluate BNCT Neutron Beams Combining Physical, Radiobiological, and Dosimetric Figures of Merit.

(1) Background:The quality of neutron beams for Boron Neutron Capture Therapy (BNCT) is currently defined by its physical characteristics in air. Recommendations exist to define whether a designed beam is useful for clinical treatment. This work presents a new way to evaluate neutron beams based on their clinical performance and on their safety, employing radiobiological quantities. (2) Methods: The case study is a neutron beam for deep-seated tumors from a 5 MeV proton beam coupled to a beryllium target. Physical Figures of Merit were used to design five beams; however, they did not allow a clear ranking of their quality in terms of therapeutic potential. The latter was then evaluated based on in-phantom dose distributions and on the calculation of the Uncomplicated Tumor Control Probability (UTCP). The safety of the beams was also evaluated calculating the in-patient out-of-beam dosimetry. (3) Results: All the beams ensured a UTCP comparable to the one of a clinical beam in phantom; the safety criterion allowed to choose the best candidate. When this was tested in the treatment planning of a real patient treated in Finland, the UTCP was still comparable to the one of the clinical beam. (4) Conclusions: Even when standard physical recommendations are not met, radiobiological and dosimetric criteria demonstrate to be a valid tool to select an effective and safe beam for patient treatment.

Colocalization of tracks from boron neutron capture reactions and images of isolated cells.

In Boron Neutron Capture Therapy, the boronated drug plays a leading role in delivering a lethal dose to the tumour. The effectiveness depends on the boron macroscopic concentration and on its distribution at sub-cellular level. This work shows a way to colocalize alpha particles and lithium ions tracks with cells. A neutron autoradiography technique is used, which combines images of cells with images of tracks produced in a solid-state nuclear track detector.

In vitro and in vivo BNCT investigations using a carborane containing sulfonamide targeting CAIX epitopes on malignant pleural mesothelioma and breast cancer cells.

This study aims at merging the therapeutic effects associated to the inhibition of Carbonic Anhydrase IX (CAIX), an essential enzyme overexpressed by cancer cells including mesothelioma and breast cancer, with those ones brought by the application of Boron Neutron Capture Therapy (BNCT). This task was pursued by designing a sulfonamido-functionalised-carborane (CA-SF) that acts simultaneously as CAIX inhibitor and boron delivery agent. The CAIX expression, measured by Western blot analysis, resulted high in both mesothelioma and breast tumours. This finding was exploited for the delivery of a therapeutic dose of boron (> 20 µg/g) to the cancer cells. The synergic cytotoxic effects operated by the enzymatic inhibition and neutron irradiation was evaluated in vitro on ZL34, AB22 and MCF7 cancer cells. Next, an in vivo model was prepared by subcutaneous injection of AB22 cells in Balb/c mice and CA-SF was administered as inclusion complex with a ß-cyclodextrin oligomer. After irradiation with thermal neutrons tumour growth was evaluated for 25 days by MRI. The obtained results appear very promising as the tumour growth was definitively markedly lower in comparison to controls and the CAIX inhibitor alone. This approach appears promising and it call consideration for the design of new therapeutic routes to cure patients affected by this disease.

Recent advances in the development of high-resolution 3D cadmium-zinc-telluride drift strip detectors.

In the last two decades, great efforts have been made in the development of 3D cadmium-zinc-telluride (CZT) detectors operating at room temperature for gamma-ray spectroscopic imaging. This work presents the spectroscopic performance of new high-resolution CZT drift strip detectors, recently developed at IMEM-CNR of Parma (Italy) in collaboration with due2lab (Italy). The detectors (19.4 mm × 19.4 mm × 6 mm) are organized into collecting anode strips (pitch of 1.6 mm) and drift strips (pitch of 0.4 mm) which are negatively biased to optimize electron charge collection. The cathode is divided into strips orthogonal to the anode strips with a pitch of 2 mm. Dedicated pulse processing analysis was performed on a wide range of collected and induced charge pulse shapes using custom 32-channel digital readout electronics. Excellent room-temperature energy resolution (1.3% FWHM at 662 keV) was achieved using the detectors without any spectral corrections. Further improvements (0.8% FWHM at 662 keV) were also obtained through a novel correction technique based on the analysis of collected-induced charge pulses from anode and drift strips. These activities are in the framework of two Italian research projects on the development of spectroscopic gamma-ray imagers (10-1000 keV) for astrophysical and medical applications.

Design of a BNCT irradiation room based on proton accelerator and beryllium target.

Preliminary studies for the design of an accelerator-based BNCT clinical facility are presented. The Beam Shaping Assembly neutron activation was evaluated experimentally and with Monte Carlo simulations. The activations of patient, air and walls in the room, the absorbed doses by the patient and the in-air dose distributions were evaluated. Based on these calculations, different walls compositions were tested to optimize the environmental conditions. Borated concrete, advantageously reducing the thermal flux in the room, was proven the best choice.

Design, synthesis and preliminary in-vitro studies of novel boronated monocarbonyl analogues of Curcumin (BMAC) for antitumor and ß-amiloyd disaggregation activity.

Curcumin is currently being investigated for its capacity to treat many types of cancer and to prevent the neuron damage that is observed in Alzheimer's disease (AD). However, its clinical use is limited by its low stability and solubility in aqueous solutions. In this study, we propose a completely new class of boronated monocarbonyl analogues of Curcumin (BMAC, 6a-c), in which a carbonyl group replaces the Curcumin ß-diketone functionality, and an ortho-carborane, an icosahedral boron cluster, substitutes one of the two phenolic rings. BMAC antitumor activity against MCF7 and OVCAR-3 cell lines was assessed in vitro and compared to that of Curcumin and the corresponding MAC derivative. BMAC 6a-c showed efficiencies that are comparable to that of MAC and superior to that of Curcumin in both the cell lines. Moreover, the inhibition of the formation of ß-amyloid aggregates by BMAC 6a-c was evaluated and it was shown that compound 6c, which contains two OH moieties, has a better efficiency than Curcumin. The presence of a second -OH group can enhance the compound's binding efficacy with ß-amyloid aggregates. For the future, the presence of at least one carborane group means that the BMAC antitumor effect can be coupled with Boron Neutron Capture Therapy.

Charged particle spectrometry to measure 10B concentration in bone.

Osteosarcoma is the most common primary malignant tumour of bone in young patients. The survival of these patients has largely been improved due to adjuvant and neo-adjuvant chemotherapy in addition to surgery. Boron neutron capture therapy (BNCT) is proposed as a complementary therapy, due to its ability to inactivate tumour cells that may survive the standard treatment and that may be responsible for recurrences and/or metastases. BNCT is based on neutron irradiation of a tumour enriched in 10B with a boron-loaded drug. Low-energy neutron capture in 10B creates charged particles that impart a high dose to tumour cells, which can be calculated only knowing the boron concentration. Charged particle spectrometry is a method that can be used to quantify boron concentration. This method requires acquisition of the energy spectra of charged particles such as alpha particles produced by neutron capture reactions in thin tissue sections irradiated with low-energy neutrons. Boron concentration is then determined knowing the stopping power of the alpha particles in the sample material. This paper describes the adaptation of this method for bone, with emphasis on sample preparation, experimental set-up and stopping power assessment of the involved alpha particles. The knowledge of boron concentration in healthy bones is important, because it allows for any dose limitation that might be necessary to avoid adverse effects such as bone fragility. The measurement process was studied through Monte Carlo simulations and analytical calculations. Finally, the boron content of bone samples was measured by alpha spectrometry at the TRIGA reactor in Pavia, Italy, and compared to that obtained by neutron autoradiography. The agreement between the results obtained with these techniques confirms the suitability of alpha spectrometry to measure boron in bone.

Translational boron neutron capture therapy (BNCT) studies for the treatment of tumors in lung.

PURPOSE: Boron neutron capture therapy (BNCT) combines selective accumulation of 10B carriers in tumor tissue with subsequent neutron irradiation. BNCT has been proposed for the treatment of multiple, non-resectable, diffuse tumors in lung. The aim of the present study was to evaluate the therapeutic efficacy and toxicity of BNCT in an experimental model of lung metastases of colon carcinoma in BDIX rats and perform complementary survival studies. MATERIALS AND METHODS: We evaluated tumor control and toxicity in lung 2 weeks post-BNCT at 2 dose levels, including 5 experimental groups per dose level: T0 (euthanized pre-treatment), Boronophenylalanine-BNCT (BPA-BNCT), BPA + Sodium decahydrodecaborate-BNCT ((BPA + GB-10)-BNCT), Beam only (BO) and Sham (no treatment, same manipulation). Tumor response was assessed employing macroscopic and microscopic end-points. An additional experiment was performed to evaluate survival and oxygen saturation in blood. RESULTS AND CONCLUSIONS: No dose-limiting signs of short/medium-term toxicity were observed in lung. All end-points revealed statistically significant BNCT-induced tumor control vs Sham at both dose levels. The survival experiment showed a statistically significant 45% increase in post-treatment survival time in the BNCT group (48 days) versus Sham (33 days). These data consistently revealed growth suppression of lung metastases by BNCT with no manifest lung toxicity. Highlights Boron Neutron Capture Therapy suppresses growth of experimental lung metastases No BNCT-induced short/medium-term toxicity in lung is associated with tumor control Boron Neutron Capture Therapy increased post-treatment survival time by 45.

An innovative therapeutic approach for malignant mesothelioma treatment based on the use of Gd/boron multimodal probes for MRI guided BNCT.

The aim of this study is to develop an innovative imaging guided approach based on Boron Neutron Capture Therapy, for the treatment of mesothelioma, assisted by the quantification of the in vivo boron distribution by MRI. The herein reported results demonstrate that overexpressed Low Density Lipoproteins receptors can be successfully exploited to deliver to mesothelioma cells a therapeutic dose of boron (26 µg/g), significantly higher than in the surrounding tissue (3.5 µg/g). Boron and Gd cells uptake was assessed by ICP-MS and MRI on two mesothelioma (ZL34, AE17) and two healthy (MRC-5 and NMuMg) cell lines. An in vivo model was prepared by subcutaneous injection of ZL34 cells in Nu/Nu mice. After irradiation with thermal neutrons, tumor growth was evaluated for 40 days by MRI. Tumor masses of boron treated mice showed a drastic reduction of about 80-85%. The obtained results appear very promising providing patients affected by this rare disease with an improved therapeutic option, exploiting LDL transporters.

Extending neutron autoradiography technique for boron concentration measurements in hard tissues.

The neutron autoradiography technique using polycarbonate nuclear track detectors (NTD) has been extended to quantify the boron concentration in hard tissues, an application of special interest in Boron Neutron Capture Therapy (BNCT). Chemical and mechanical processing methods to prepare thin tissue sections as required by this technique have been explored. Four different decalcification methods governed by slow and fast kinetics were tested in boron-loaded bones. Due to the significant loss of the boron content, this technique was discarded. On the contrary, mechanical manipulation to obtain bone powder and tissue sections of tens of microns thick proved reproducible and suitable, ensuring a proper conservation of the boron content in the samples. A calibration curve that relates the 10B concentration of a bone sample and the track density in a Lexan NTD is presented. Bone powder embedded in boric acid solution with known boron concentrations between 0 and 100 ppm was used as a standard material. The samples, contained in slim Lexan cases, were exposed to a neutron fluence of 1012 cm-2 at the thermal column central facility of the RA-3 reactor (Argentina). The revealed tracks in the NTD were counted with an image processing software. The effect of track overlapping was studied and corresponding corrections were implemented in the presented calibration curve. Stochastic simulations of the track densities produced by the products of the 10B thermal neutron capture reaction for different boron concentrations in bone were performed and compared with the experimental results. The remarkable agreement between the two curves suggested the suitability of the obtained experimental calibration curve. This neutron autoradiography technique was finally applied to determine the boron concentration in pulverized and compact bone samples coming from a sheep experimental model. The obtained results for both type of samples agreed with boron measurements carried out by ICP-OES within experimental uncertainties. The fact that the histological structure of bone sections remains preserved allows for future boron microdistribution analysis.