The application of metal artifact reduction methods on computed tomography scans for radiotherapy applications: A literature review.

Metal artifact reduction (MAR) methods are used to reduce artifacts from metals or metal components in computed tomography (CT). In radiotherapy (RT), CT is the most used imaging modality for planning, whose quality is often affected by metal artifacts. The aim of this study is to systematically review the impact of MAR methods on CT Hounsfield Unit values, contouring of regions of interest, and dose calculation for RT applications. This systematic review is performed in accordance with the PRISMA guidelines; the PubMed and Web of Science databases were searched using the main keywords "metal artifact reduction", "computed tomography" and "radiotherapy". A total of 382 publications were identified, of which 40 (including one review article) met the inclusion criteria and were included in this review. The selected publications (except for the review article) were grouped into two main categories: commercial MAR methods and research-based MAR methods. Conclusion: The application of MAR methods on CT scans can improve treatment planning quality in RT. However, none of the investigated or proposed MAR methods was completely satisfactory for RT applications because of limitations such as the introduction of other errors (e.g., other artifacts) or image quality degradation (e.g., blurring), and further research is still necessary to overcome these challenges.

An innovative intramedullary bone graft harvesting concept as a fundamental component of scaffold-guided bone regeneration: A preclinical in vivo validation.

Background: The deployment of bone grafts (BGs) is critical to the success of scaffold-guided bone regeneration (SGBR) of large bone defects. It is thus critical to provide harvesting devices that maximize osteogenic capacity of the autograft while also minimizing graft damage during collection. As an alternative to the Reamer-Irrigator-Aspirator 2 (RIA 2) system - the gold standard for large-volume graft harvesting used in orthopaedic clinics today - a novel intramedullary BG harvesting concept has been preclinically introduced and referred to as the ARA (aspirator + reaming-aspiration) concept. The ARA concept uses aspiration of the intramedullary content, followed by medullary reaming-aspiration of the endosteal bone. This concept allows greater customization of BG harvesting conditions vis-à-vis the RIA 2 system. Following its successful in vitro validation, we hypothesized that an ARA concept-collected BG would have comparable in vivo osteogenic capacity compared to the RIA 2 system-collected BG. Methods: We used 3D-printed, medical-grade polycaprolactone-hydroxyapatite (mPCL-HA, wt 96 %:4 %) scaffolds with a Voronoi design, loaded with or without different sheep-harvested BGs and tested them in an ectopic bone formation rat model for up to 8 weeks. Results: Active bone regeneration was observed throughout the scaffold-BG constructs, particularly on the surface of the bone chips with endochondral bone formation, and highly vascularized tissue formed within the fully interconnected pore architecture. There were no differences between the BGs derived from the RIA 2 system and the ARA concept in new bone volume formation and in compression tests (Young's modulus, p = 0.74; yield strength, p = 0.50). These results highlight that the osteogenic capacities of the mPCL-HA Voronoi scaffold loaded with BGs from the ARA concept and the RIA 2 system are equivalent. Conclusion: In conclusion, the ARA concept offers a promising alternative to the RIA 2 system for harvesting BGs to be clinically integrated into SGBR strategies. The translational potential of this article: Our results show that biodegradable composite scaffolds loaded with BGs from the novel intramedullary harvesting concept and the RIA 2 system have equivalent osteogenic capacity. Thus, the innovative, highly intuitive intramedullary harvesting concept offers a promising alternative to the RIA 2 system for harvesting bone grafts, which are an important component for the routine translation of SGBR concepts into clinical practice.

Deep learning-based ultrasound transducer induced CT metal artifact reduction using generative adversarial networks for ultrasound-guided cardiac radioablation.

In US-guided cardiac radioablation, a possible workflow includes simultaneous US and planning CT acquisitions, which can result in US transducer-induced metal artifacts on the planning CT scans. To reduce the impact of these artifacts, a metal artifact reduction (MAR) algorithm has been developed based on a deep learning Generative Adversarial Network called Cycle-MAR, and compared with iMAR (Siemens), O-MAR (Philips) and MDT (ReVision Radiology), and CCS-MAR (Combined Clustered Scan-based MAR). Cycle-MAR was trained with a supervised learning scheme using sets of paired clinical CT scans with and without simulated artifacts. It was then evaluated on CT scans with real artifacts of an anthropomorphic phantom, and on sets of clinical CT scans with simulated artifacts which were not used for Cycle-MAR training. Image quality metrics and HU value-based analysis were used to evaluate the performance of Cycle-MAR compared to the other algorithms. The proposed Cycle-MAR network effectively reduces the negative impact of the metal artifacts. For example, the calculated HU value improvement percentage for the cardiac structures in the clinical CT scans was 59.58%, 62.22%, and 72.84% after MDT, CCS-MAR, and Cycle-MAR application, respectively. The application of MAR algorithms reduces the impact of US transducer-induced metal artifacts on CT scans. In comparison to iMAR, O-MAR, MDT, and CCS-MAR, the application of developed Cycle-MAR network on CT scans performs better in reducing these metal artifacts.

Shear wave velocity measurement of the placenta is not limited by placental location.

INTRODUCTION: Ultrasound elastography shows diagnostic promise via the non-invasive determination of placental elastic properties. A limitation is a potential for inadequate measurements from posterior placentae. This study aimed to analyse placental position's influence on measures of shear wave elastography (SWV). METHODS: SWV elastography measurements were obtained via ultrasound at 24, 28 and 36 weeks gestation from 238 pregnancies. . The placental position was labelled as either anterior, posterior or fundal/lateral. Average SWV measurements (m/s) and the corresponding standard deviations (SD) were used for data analysis. RESULTS: There was a statistically significant difference between SWV recorded from anterior (1.33 ± 0.19)m/s and posterior (1.39 ± 0.18)m/s placentae (p < 0.001). However, the average sampling depth between these groups was significantly different (3.98 cm vs. 5.38 cm, p < 0.001). There was no statistically significant difference between SWV when measurements were compared at similar depths, regardless of placental location. The addition of placental position to a previously developed mixed-effects model confirmed placental position did not result in improved SWV measurements. In this model, sampling depth remained the best predictor for SWV. CONCLUSIONS: This study showed that placental position does not influence the accuracy or reliability of SWV.

Protocol for the BONE-RECON trial: a single-arm feasibility trial for critical sized lower limb BONE defect RECONstruction using the mPCL-TCP scaffold system with autologous vascularised corticoperiosteal tissue transfer.

INTRODUCTION: Reconstruction of critical bone defects is challenging. In a substantial subgroup of patients, conventional reconstructive techniques are insufficient. Biodegradable scaffolds have emerged as a novel tissue engineering strategy for critical-sized bone defect reconstruction. A corticoperiosteal flap integrates the hosts' ability to regenerate bone and permits the creation of a vascular axis for scaffold neo-vascularisation (regenerative matching axial vascularisation-RMAV). This phase IIa study evaluates the application of the RMAV approach alongside a custom medical-grade polycaprolactone-tricalcium phosphate (mPCL-TCP) scaffold (Osteopore) to regenerate bone sufficient to heal critical size defects in lower limb defects. METHODS AND ANALYSIS: This open-label, single-arm feasibility trial will be jointly coordinated by the Complex Lower Limb Clinic (CLLC) at the Princess Alexandra Hospital in Woolloongabba (Queensland, Australia), the Australian Centre for Complex Integrated Surgical Solutions (Queensland, Australia) and the Faculty of Engineering, Queensland University of Technology in Kelvin Grove (Queensland, Australia). Aiming for limb salvage, the study population (n=10) includes any patient referred to the CLLC with a critical-sized bone defect not amenable to conventional reconstructive approaches, after discussion by the interdisciplinary team. All patients will receive treatment using the RMAV approach using a custom mPCL-TCP implant. The primary study endpoint will be safety and tolerability of the reconstruction. Secondary end points include time to bone union and weight-bearing status on the treated limb. Results of this trial will help shape the role of scaffold-guided bone regenerative approaches in complex lower limb reconstruction where current options remain limited. ETHICS AND DISSEMINATION: Approval was obtained from the Human Research Ethics Committee at the participating centre. Results will be submitted for publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: ACTRN12620001007921.

In vivo characterization of 3D-printed polycaprolactone-hydroxyapatite scaffolds with Voronoi design to advance the concept of scaffold-guided bone regeneration.

Three-dimensional (3D)-printed medical-grade polycaprolactone (mPCL) composite scaffolds have been the first to enable the concept of scaffold-guided bone regeneration (SGBR) from bench to bedside. However, advances in 3D printing technologies now promise next-generation scaffolds such as those with Voronoi tessellation. We hypothesized that the combination of a Voronoi design, applied for the first time to 3D-printed mPCL and ceramic fillers (here hydroxyapatite, HA), would allow slow degradation and high osteogenicity needed to regenerate bone tissue and enhance regenerative properties when mixed with xenograft material. We tested this hypothesis in vitro and in vivo using 3D-printed composite mPCL-HA scaffolds (wt 96%:4%) with the Voronoi design using an ISO 13485 certified additive manufacturing platform. The resulting scaffold porosity was 73% and minimal in vitro degradation (mass loss <1%) was observed over the period of 6 months. After loading the scaffolds with different types of fresh sheep xenograft and ectopic implantation in rats for 8 weeks, highly vascularized tissue without extensive fibrous encapsulation was found in all mPCL-HA Voronoi scaffolds and endochondral bone formation was observed, with no adverse host-tissue reactions. This study supports the use of mPCL-HA Voronoi scaffolds for further testing in future large preclinical animal studies prior to clinical trials to ultimately successfully advance the SGBR concept.

An in vivo study to investigate an original intramedullary bone graft harvesting technology.

BACKGROUND: Harvesting bone graft (BG) from the intramedullary canal to treat bone defects is largely conducted using the Reamer-Irrigator-Aspirator (RIA) system. The RIA system uses irrigation fluid during harvesting, which may result in washout of osteoinductive factors. Here, we propose a new harvesting technology dedicated to improving BG collection without the potential washout effect of osteoinductive factors associated with irrigation fluid. This novel technology involves the conceptual approach of first aspirating the bone marrow (BM) with a novel aspirator prototype, followed by reaming with standard reamers and collecting the bone chips with the aspirator (reaming-aspiration method, R-A method). The aim of this study was to assess the harvesting efficacy and osteoinductive profile of the BG harvested with RIA 2 system (RIA 2 group) compared to the novel harvesting concept (aspirator + R-A method, ARA group). METHODS: Pre-planning computed tomography (CT) imaging was conducted on 16 sheep to determine the femoral isthmus canal diameter. In this non-recovery study, sheep were divided into two groups: RIA 2 group (n = 8) and ARA group (n = 8). We measured BG weight collected from left femur and determined femoral cortical bone volume reduction in postoperative CT imaging. Growth factor and inflammatory cytokine amounts of the BGs were quantified using enzyme-linked immunosorbent assay (ELISA) methods. RESULTS: The use of the stand-alone novel aspirator in BM collection, and in harvesting BG when the aspirator is used in conjunction with sequential reaming (R-A method) was proven feasible. ELISA results showed that the collected BG contained relevant amounts of growth factors and inflammatory cytokines in both the RIA 2 and the ARA group. CONCLUSIONS: Here, we present the first results of an innovative concept for harvesting intramedullary BG. It is a prototype of a novel aspirator technology that enables the stepwise harvesting of first BM and subsequent bone chips from the intramedullary canal of long bones. Both the BG collected with the RIA 2 system and the aspirator prototype had the capacity to preserve the BG's osteoinductive microenvironment. Future in vivo studies are required to confirm the bone regenerative capacity of BG harvested with the innovative harvesting technology.

Convergence of scaffold-guided bone regeneration principles and microvascular tissue transfer surgery.

A preclinical evaluation using a regenerative medicine methodology comprising an additively manufactured medical-grade ε-polycaprolactone ß-tricalcium phosphate (mPCL-TCP) scaffold with a corticoperiosteal flap was undertaken in eight sheep with a tibial critical-size segmental bone defect (9.5 cm3, M size) using the regenerative matching axial vascularization (RMAV) approach. Biomechanical, radiological, histological, and immunohistochemical analysis confirmed functional bone regeneration comparable to a clinical gold standard control (autologous bone graft) and was superior to a scaffold control group (mPCL-TCP only). Affirmative bone regeneration results from a pilot study using an XL size defect volume (19 cm3) subsequently supported clinical translation. A 27-year-old adult male underwent reconstruction of a 36-cm near-total intercalary tibial defect secondary to osteomyelitis using the RMAV approach. Robust bone regeneration led to complete independent weight bearing within 24 months. This article demonstrates the widely advocated and seldomly accomplished concept of "bench-to-bedside" research and has weighty implications for reconstructive surgery and regenerative medicine more generally.

Combined clustered scan-based metal artifact reduction algorithm (CCS-MAR) for ultrasound-guided cardiac radioablation.

Cardiac radioablation is a promising treatment for cardiac arrhythmias, but accurate dose delivery can be affected by heart motion. For this reason, real-time cardiac motion monitoring during radioablation is of paramount importance. Real-time ultrasound (US) guidance can be a solution. The US-guided cardiac radioablation workflow can be simplified by the simultaneous US and planning computed tomography (CT) acquisition, which can result in US transducer-induced metal artifacts on the planning CT scans. To reduce the impact of these artifacts, a new metal artifact reduction (MAR) algorithm (named: Combined Clustered Scan-based MAR [CCS-MAR]) has been developed and compared with iMAR (Siemens), O-MAR (Philips) and MDT (ReVision Radiology) algorithms. CCS-MAR is a fully automated sinogram inpainting-based MAR algorithm, which uses a two-stage correction process based on a normalized MAR method. The second stage aims to correct errors remaining from the first stage to create an artifact-free combined clustered scan for the process of metal artifact reduction. To evaluate the robustness of CCS-MAR, conventional CT scans and/or dual-energy CT scans from three anthropomorphic phantoms and transducers with different sizes were used. The performance of CCS-MAR for metal artifact reduction was compared with other algorithms through visual comparison, image quality metrics analysis, and HU value restoration evaluation. The results of this study show that CCS-MAR effectively reduced the US transducer-induced metal artifacts and that it improved HU value accuracy more or comparably to other MAR algorithms. These promising results justify future research into US transducer-induced metal artifact reduction for the US-guided cardiac radioablation purposes.

Bone Regeneration Exploiting Corticoperiosteal Tissue Transfer for Scaffold-Guided Bone Regeneration.

Contemporary reconstructive approaches for critical size bone defects carry significant disadvantages. As a result, clinically driven research has focused on the development and translation of alternative therapeutic concepts. Scaffold-guided tissue regeneration (SGTR) is an emerging technique to heal critical size bone defects. However, issues synchronizing scaffold vascularization with bone-specific regenerative processes currently limit bone regeneration for extra large (XL, 19 cm3) critical bone defects. To address this issue, we developed a large animal model that incorporates a corticoperiosteal flap (CPF) for sustained scaffold neovascularization and bone regeneration. In 10 sheep, we demonstrated the efficacy of this approach for healing medium (M, 9 cm3) size critical bone defects as demonstrated on plain radiography, microcomputed tomography, and histology. Furthermore, in two sheep, we demonstrate how this approach can be safely extended to heal XL critical size defects. This article presents an original CPF technique in a well-described preclinical model, which can be used in conjunction with the SGTR concept, to address challenging critical size bone defects in vivo. Impact statement This article describes a novel scaffold-guided tissue engineering approach utilizing a corticoperiosteal flap for bone healing in critical size long bone defects. This approach will be of use for tissue engineers and surgeons exploring vascularized tissue transfer as an option to regenerate large volumes of bone for extensive critical size bone defects both in vivo and in the clinical arena.

Clinical translation of a patient-specific scaffold-guided bone regeneration concept in four cases with large long bone defects.

Background: Bone defects after trauma, infection, or tumour resection present a challenge for patients and clinicians. To date, autologous bone graft (ABG) is the gold standard for bone regeneration. To address the limitations of ABG such as limited harvest volume as well as overly fast remodelling and resorption, a new treatment strategy of scaffold-guided bone regeneration (SGBR) was developed. In a well-characterized sheep model of large to extra-large tibial segmental defects, three-dimensional (3D) printed composite scaffolds have shown clinically relevant biocompatibility and osteoconductive capacity in SGBR strategies. Here, we report four challenging clinical cases with large complex posttraumatic long bone defects using patient-specific SGBR as a successful treatment. Methods: After giving informed consent computed tomography (CT) images were used to design patient-specific biodegradable medical-grade polycaprolactone-tricalcium phosphate (mPCL-TCP, 80:20 â€‹wt%) scaffolds. The CT scans were segmented using Materialise Mimics to produce a defect model and the scaffold parts were designed with Autodesk Meshmixer. Scaffold prototypes were 3D-printed to validate robust clinical handling and bone defect fit. The final scaffold design was additively manufactured under Food and Drug Administration (FDA) guidelines for patient-specific and custom-made implants by Osteopore International Pte Ltd. Results: Four patients (age: 23-42 years) with posttraumatic lower extremity large long bone defects (case 1: 4 â€‹cm distal femur, case 2: 10 â€‹cm tibia shaft, case 3: complex malunion femur, case 4: irregularly shaped defect distal tibia) are presented. After giving informed consent, the patients were treated surgically by implanting a custom-made mPCL-TCP scaffold loaded with ABG (case 2: additional application of recombinant human bone morphogenetic protein-2) harvested with the Reamer-Irrigator-Aspirator system (RIA, Synthes®). In all cases, the scaffolds matched the actual anatomical defect well and no perioperative adverse events were observed. Cases 1, 3 and 4 showed evidence of bony ingrowth into the large honeycomb pores (pores >2 â€‹mm) and fully interconnected scaffold architecture with indicative osseous bridges at the bony ends on the last radiographic follow-up (8-9 months after implantation). Comprehensive bone regeneration and full weight bearing were achieved in case 2 â€‹at follow-up 23 months after implantation. Conclusion: This study shows the bench to bedside translation of guided bone regeneration principles into scaffold-based bone tissue engineering. The scaffold design in SGBR should have a tissue-specific morphological signature which stimulates and directs the stages from the initial host response towards the full regeneration. Thereby, the scaffolds provide a physical niche with morphology and biomaterial properties that allow cell migration, proliferation, and formation of vascularized tissue in the first one to two months, followed by functional bone formation and the capacity for physiological bone remodelling. Great design flexibility of composite scaffolds to support the one to three-year bone regeneration was observed in four patients with complex long bone defects. The translational potential of this article: This study reports on the clinical efficacy of SGBR in the treatment of long bone defects. Moreover, it presents a comprehensive narrative of the rationale of this technology, highlighting its potential for bone regeneration treatment regimens in patients with any type of large and complex osseous defects.

Regenerative matching axial vascularisation of absorbable 3D-printed scaffold for large bone defects: A first in human series.

BACKGROUND: We describe the first clinical series of a novel bone replacement technique based on regenerative matching axial vascularisation (RMAV). This was used in four cases: a tibial defect after treatment of osteomyelitis; a calvarial defect after trauma and failed titanium cranioplasty; a paediatric tibial defect after neoadjuvant chemotherapy and resection of Ewing sarcoma; and a paediatric mandibular deficiency resulting from congenital hemifacial microsomia. METHOD: All patients underwent reconstruction with three-dimensional (3D)-printed medical-grade polycaprolactone and tricalcium phosphate (mPCL-TCP) scaffolds wrapped in vascularised free corticoperiosteal flaps. OUTCOME: Functional volumes of load-sharing regenerate bone have formed in all cases after a moderate duration of follow-up. At 36 cm, case 1 remains the longest segment of load bearing bone ever successfully reconstructed. This technique offers an alternative to existing methods of large volume bone defect reconstruction that may be safe, reliable, and give predictable outcomes in challenging situations. It achieves this by using a bioresorbable scaffold to support and direct the growth of regenerate bone, driven by RMAV. CONCLUSION: This technique may facilitate the reconstruction of bone defects previously thought unreconstructable, reduce the risk of long-term implant-related complications and achieve these outcomes in a hostile environment. These potential benefits must now be formally tested in prospective clinical trials.

Changes in placental elastography in the third trimester - Analysis using a linear mixed effect model.

INTRODUCTION: Research into the role of ultrasound elastography to assess compromised placental tissue is ongoing. There is particular interest in evaluating its potential in the investigation of changes associated with uteroplacental dysfunction. To date, there is limited data on how different maternal and fetal considerations, such as advancing gestational age, amniotic fluid Index (AFI) and maternal body mass index (BMI) may influence shear wave velocity (SWV) measurements. This study aimed to evaluate longitudinal changes in SWV throughout gestation and model these changes with other developing fetal and maternal physiological and biological characteristics. METHODS: The study utilised 238 singleton pregnancies and collected longitudinal data at repeated intervals in the 3rd trimester representing 629 individual data points. Linear mixed model regression analysis was used to identify significant predictors for SWV. RESULTS: From a total of ten variables selected for modelling, only gestational age, AFI, BMI, and sample depth were found to be significant predictors of placental SWV, and gestational age and AFI were found to have only a minimal impact on SWV. DISCUSSION: Sophisticated statistical modelling demonstrates that many of the expected maternal and fetal changes in the 3rd trimester have no or minimal impact on placental SWV. Understanding which factors influence placental SWV is essential to ascertain the technique's utility in managing pregnancies complicated by placental dysfunction in the future.

In vitro engineering of a bone metastases model allows for study of the effects of antiandrogen therapies in advanced prostate cancer.

While androgen-targeted therapies are routinely used in advanced prostate cancer (PCa), their effect is poorly understood in treating bone metastatic lesions and ultimately results in the development of metastatic castrate resistant prostate cancer (mCRPC). Here, we used an all-human microtissue-engineered model of mineralized metastatic tissue combining human osteoprogenitor cells, 3D printing and prostate cancer cells, to assess the effects of the antiandrogens, bicalutamide, and enzalutamide in this microenvironment. We demonstrate that cancer/bone stroma interactions and antiandrogens drive cancer progression in a mineralized microenvironment. Probing the bone microenvironment with enzalutamide led to stronger cancer cell adaptive responses and osteomimicry than bicalutamide. Enzalutamide presented with better treatment response, in line with enzalutamide delaying time to bone-related events and enzalutamide extending survival in mCRPC. The all-human microtissue-engineered model of mineralized metastatic tissue presented here represents a substantial advance to dissect the role of the bone tumor microenvironment and responses to therapies for mCPRC.

A New Automated Histomorphometric MATLAB Algorithm for Immunohistochemistry Analysis Using Whole Slide Imaging.

The use of animal models along with the employment of advanced and sophisticated stereological methods for assessing bone quality combined with the use of statistical methods to evaluate the effectiveness of bone therapies has made it possible to investigate the pathways that regulate bone responses to medical devices. Image analysis of histomorphometric measurements remains a time-consuming task, as the image analysis software currently available does not allow for automated image segmentation. Such a feature is usually obtained by machine learning and with software platforms that provide image-processing tools such as MATLAB. In this study, we introduce a new MATLAB algorithm to quantify immunohistochemically stained critical-sized bone defect samples and compare the results with the commonly available Aperio Image Scope Positive Pixel Count (PPC) algorithm. Bland and Altman analysis and Pearson correlation showed that the measurements acquired with the new MATLAB algorithm were in excellent agreement with the measurements obtained with the Aperio PPC algorithm, and no significant differences were found within the histomorphometric measurements. The ability to segment whole slide images, as well as defining the size and the number of regions of interest to be quantified, makes this MATLAB algorithm a potential histomorphometric tool for obtaining more objective, precise, and reproducible quantitative assessments of entire critical-sized bone defect image data sets in an efficient and manageable workflow.

Histomorphometric Evaluation of Critical-Sized Bone Defects Using Osteomeasure and Aperio Image Analysis Systems.

Most histological evaluations of critical-sized bone defects are limited to the analysis of a few regions of interest at a time. Manual and semiautomated histomorphometric approaches often have intra- and interobserver subjectivity, as well as variability in image analysis methods. Moreover, the production of large image data sets makes histological assessment and histomorphometric analysis labor intensive and time consuming. Herein, we tested and compared two image segmentation methods: thresholding (automated) and region-based (manual) modes, for quantifying complete image sets across entire critical-sized bone defects, using the widely used Osteomeasure system and the freely downloadable Aperio Image Scope software. A comparison of bone histomorphometric data showed strong agreement between the automated segmentation mode of the Osteomeasure software with the manual segmentation mode of Aperio Image Scope analysis (bone formation R2 = 0.9615 and fibrous tissue formation R2 = 0.8734). These results indicate that Aperio is capable of handling large histological images, with excellent speed performance in producing highly consistent histomorphometric evaluations compared with the Osteomeasure image analysis system. The statistical evaluation of these two major bone parameters demonstrated that Aperio Image Scope is as capable as Osteomeasure. This study developed a protocol to improve the quality of results and reduce analysis time, while also promoting the standardization of image analysis protocols for the histomorphometric analysis of critical-sized bone defect samples. Impact Statement Despite bone tissue engineering innovations increasing over the last decade, histomorphometric analysis of large bone defects used to study such approaches continues to pose a challenge for pathological assessment. This is due to the resulting large image data set, and the lack of a gold standard image analysis protocol to quantify histological outcomes. Herein, we present a standardized protocol for the image analysis of critical-sized bone defect samples stained with Goldner's Trichrome using the Osteomeasure and Aperio Image Scope image analysis systems. The results were critically examined to determine their reproducibility and accuracy for analyzing large bone defects.

Ultrasound-triggered release from metal shell microcapsules.

Metal shell microcapsules have been shown to completely retain their core until its release is triggered, making them a promising candidate for use as a controllable drug delivery vehicle due to their superior retention properties as compared to polymer shell microcapsules. Focused ultrasound (FUS) has been successfully utilised to trigger release of lipophilic drugs from polymer microcapsules, and in this work the response of gold shell microcapsules with and without an inner polymeric shell, to FUS and standard ultrasound is explored. The results show that gold shell microcapsules with an inner polymer shell rupture when exposed to standard ultrasound and that there is a linear correlation between the gold shell thickness and the extent of shell rupture. When FUS is applied to these microcapsules, powers as low as 0.16 W delivered in bursts of 10 ms/s over a period of 120 s are sufficient to cause rupture of 53 nm gold shell microcapsules. Additional findings suggest that gold shell microcapsules without the polymer layer dispersed in a hydrogel matrix, as opposed to aqueous media, rupture more efficiently when exposed to FUS, and that thicker gold shells are more responsive to ultrasound-triggered rupture regardless of the external environment. Release of dye from all successfully ruptured capsules was sustained over a period of between 7 and 35 days. These findings suggest that emulsion-templated gold shell microcapsules embedded in a hydrogel matrix would be suitable for use as an implantable drug delivery vehicle with FUS used to externally trigger release.

Microenvironment engineering of osteoblastic bone metastases reveals osteomimicry of patient-derived prostate cancer xenografts.

Representative in vitro models that mimic the native bone tumor microenvironment are warranted to support the development of more successful treatments for bone metastases. Here, we have developed a primary cell 3D model consisting of a human osteoblast-derived tissue-engineered construct (hOTEC) indirectly co-cultured with patient-derived prostate cancer xenografts (PDXs), in order to study molecular interactions in a patient-derived microenvironment context. The engineered biomimetic microenvironment had high mineralization and embedded osteocytes, and supported a high degree of cancer cell osteomimicry at the gene, protein and mineralization levels when co-cultured with prostate cancer PDXs from a lymph node metastasis (LuCaP35) and bone metastasis (BM18) from patients with primary prostate cancer. This fully patient-derived model is a promising tool for the assessment of new molecular mechanisms and as a personalized pre-clinical platform for therapy testing for patients with prostate cancer bone metastases.

Bone volume fraction and structural parameters for estimation of mechanical stiffness and failure load of human cancellous bone samples; in-vitro comparison of ultrasound transit time spectroscopy and X-ray µCT.

Conventional mechanical testing is the 'gold standard' for assessing the stiffness (N mm-1) and strength (MPa) of bone, although it is not applicable in-vivo since it is inherently invasive and destructive. The mechanical integrity of a bone is determined by its quantity and quality; being related primarily to bone density and structure respectively. Several non-destructive, non-invasive, in-vivo techniques have been developed and clinically implemented to estimate bone density, both areal (dual-energy X-ray absorptiometry (DXA)) and volumetric (quantitative computed tomography (QCT)). Quantitative ultrasound (QUS) parameters of velocity and attenuation are dependent upon both bone quantity and bone quality, although it has not been possible to date to transpose one particular QUS parameter into separate estimates of quantity and quality. It has recently been shown that ultrasound transit time spectroscopy (UTTS) may provide an accurate estimate of bone density and hence quantity. We hypothesised that UTTS also has the potential to provide an estimate of bone structure and hence quality. In this in-vitro study, 16 human femoral bone samples were tested utilising three techniques; UTTS, micro computed tomography (µCT), and mechanical testing. UTTS was utilised to estimate bone volume fraction (BV/TV) and two novel structural parameters, inter-quartile range of the derived transit time (UTTS-IQR) and the transit time of maximum proportion of sonic-rays (TTMP). µCT was utilised to derive BV/TV along with several bone structure parameters. A destructive mechanical test was utilised to measure the stiffness and strength (failure load) of the bone samples. BV/TV was calculated from the derived transit time spectrum (TTS); the correlation coefficient (R2) with µCT-BV/TV was 0.885. For predicting mechanical stiffness and strength, BV/TV derived by both µCT and UTTS provided the strongest correlation with mechanical stiffness (R2=0.567 and 0.618 respectively) and mechanical strength (R2=0.747 and 0.736 respectively). When respective structural parameters were incorporated to BV/TV, multiple regression analysis indicated that none of the µCT histomorphometric parameters could improve the prediction of mechanical stiffness and strength, while for UTTS, adding TTMP to BV/TV increased the prediction of mechanical stiffness to R2=0.711 and strength to R2=0.827. It is therefore envisaged that UTTS may have the ability to estimate BV/TV along with providing an improved prediction of osteoporotic fracture risk, within routine clinical practice in the future.

The dependence of broadband ultrasound attenuation on phase interference in thin plates of variable thickness and curvature: a comparison of experimental measurement and computer simulation.

The measurement of broadband ultrasound attenuation describes the linear increase in ultrasound attenuation with frequency (dB/MHz); this is generally performed at the calcaneus, consisting of a high proportion of metabolically active cancellous bone. Although broadband ultrasound attenuation is not routinely implemented within clinical management since it cannot provide a reliable estimation of bone mineral density and hence clinical definition of osteopenia and osteoporosis, it offers a reliable means to predict osteoporotic fracture risk. One of the potential factors that can influence the accuracy of broadband ultrasound attenuation measurement is the effect of cortical end plates. This study aimed to explore this, performing a comparison of experimental study and computer simulation prediction. A total of three categories of thin discs were three-dimensional (3D) printed to replicate cortical shells of (1) variable constant thickness (planar), (2) variable constant thickness (curved), and (3) variable thickness. A through-transmission technique was used, where two single-element, unfocused, 1 MHz broadband transducers, as utilised clinically, were positioned coaxially in a cylindrical holder and immersed in water. Both quantitative and qualitative analyses demonstrated that broadband ultrasound attenuation measurements of the 'planar' and 'curved' discs were not statistically different (p-values > 0.01). A cyclic relationship between broadband ultrasound attenuation and disc thickness was observed; this was replicated within a computer simulation of phase interference created by a double-reflection echo within each disc (R2 = 97.0%). Variable-thickness discs provided broadband ultrasound attenuation measurements ranging between 31.6 ± 0.1 and 40.60 ± 0.1 dB/MHz. Again applying the double-reflection echo simulation, a high level of agreement between experimental and simulation was recorded (R2 = 93.4%). This study indicates that the cortical end plate can significantly affect the broadband ultrasound attenuation measurement of cancellous bone as a result of phase interference and, therefore, warrants further investigation to minimise its effect on clinical assessment.