A multi-modal exploration of heterogeneous physico-chemical properties of DCIS breast microcalcifications.

Ductal carcinoma in situ (DCIS) is frequently associated with breast calcification. This study combines multiple analytical techniques to investigate the heterogeneity of these calcifications at the micrometre scale. X-ray diffraction, scanning electron microscopy and Raman and Fourier-transform infrared spectroscopy were used to determine the physicochemical and crystallographic properties of type II breast calcifications located in formalin fixed paraffin embedded DCIS breast tissue samples. Multiple calcium phosphate phases were identified across the calcifications, distributed in different patterns. Hydroxyapatite was the dominant mineral, with magnesium whitlockite found at the calcification edge. Amorphous calcium phosphate and octacalcium phosphate were also identified close to the calcification edge at the apparent mineral/matrix barrier. Crystallographic features of hydroxyapatite also varied across the calcifications, with higher crystallinity centrally, and highest carbonate substitution at the calcification edge. Protein was also differentially distributed across the calcification and the surrounding soft tissue, with collagen and ß-pleated protein features present to differing extents. Combination of analytical techniques in this study was essential to understand the heterogeneity of breast calcifications and how this may link crystallographic and physicochemical properties of calcifications to the surrounding tissue microenvironment.

Establishing a minimum PMI for bone sun bleaching in a UK environment with a controlled desert-simulated comparison.

Microenvironments play a significant part in understanding the post-mortem interval in forensic taphonomy. Recently, the value of weathering factors in relation to obtaining a PMI has been investigated further. In this study, observations were made to calculate the length of time it takes for three different bone elements (femur, rib, and scapula) to bleach in a UK summer and winter. This research also investigated whether there were any physicochemical modifications to the bone caused by bleaching. Porcine femora, scapulae, and ribs were placed into open and shaded areas of an outdoor research facility located in Oxfordshire, UK, during summer (July-Sep) and winter months (Dec-Mar). The specimens were monitored at 3-week intervals using photography, and an observational scoring method was developed to quantify the extent of bleaching. As temperatures are typically much lower in the UK compared with warmer climates, a controlled indoor-simulated desert experiment was also undertaken to be used as a control. This allowed sun bleaching and changes to the bone chemistry to be monitored in a controlled, high-UV environment for comparison with the UK outdoor experiments. Fourier transform infrared spectroscopy (FTIR) was employed to analyze physicochemical modifications to both the mineral and organic components of the bone. The FTIR was used to calculate crystallinity index (CI), mineral to organic ratio, and the relative amount of carbonate concentrations. Weather data was collected and a positive correlation was found between both ultraviolet (UV) levels and accumulated degree days (ADD) when compared with observational bleaching scores. Bleaching (whitening) of the bone samples occurred in both seasons but at different rates, with the bleaching process occurring at a slower rate in winter. During summer, the initial bleaching process was evident at 6 weeks, and by 9 weeks, the bones were an off-white colour. During the winter period, whitening of the bone started at 9 weeks; however, only the scapula and rib samples displayed a similar off-white colour. This colouration was observed at 13 weeks rather than at 9 weeks. The desert simulation samples started bleaching in a similar pattern to the outdoor samples after 1 week but the bones did not fully bleach. The bone chemistry, based on physicochemical properties obtained from the FTIR, showed a significant statistical difference between the simulated desert and winter season when compared against a control sample. For the winter samples, the mineral to organic ratio was significantly higher than that in the control, suggesting a reduction in the proportion of organic. For the samples in the simulated desert environment, the crystallinity index was significantly higher than that in the control samples, suggesting an increase in crystallinity. The results of this experiment support the fact that it is possible to achieve bleaching in a UK environment and that the minimal time frame for this to occur differs in seasons.

Calcification Microstructure Reflects Breast Tissue Microenvironment.

Microcalcifications are important diagnostic indicators of disease in breast tissue. Tissue microenvironments differ in many aspects between normal and cancerous cells, notably extracellular pH and glycolytic respiration. Hydroxyapatite microcalcification microstructure is also found to differ between tissue pathologies, including differential ion substitutions and the presence of additional crystallographic phases. Distinguishing between tissue pathologies at an early stage is essential to improve patient experience and diagnostic accuracy, leading to better disease outcome. This study explores the hypothesis that microenvironment features may become immortalised within calcification crystallite characteristics thus becoming indicators of tissue pathology. In total, 55 breast calcifications incorporating 3 tissue pathologies (benign - B2, ductal carcinoma in-situ - B5a and invasive malignancy - B5b) from archive formalin-fixed paraffin-embedded core needle breast biopsies were analysed using X-ray diffraction. Crystallite size and strain were determined from 548 diffractograms using Williamson-Hall analysis. There was an increased crystallinity of hydroxyapatite with tissue malignancy compared to benign tissue. Coherence length was significantly correlated with pathology grade in all basis crystallographic directions (P < 0.01), with a greater difference between benign and in situ disease compared to in-situ disease and invasive malignancy. Crystallite size and non-uniform strain contributed to peak broadening in all three pathologies. Furthermore, crystallite size and non-uniform strain normal to the basal planes increased significantly with malignancy (P < 0.05). Our findings support the view that tissue microenvironments can influence differing formation mechanisms of hydroxyapatite through acidic precursors, leading to differential substitution of carbonate into the hydroxide and phosphate sites, causing significant changes in crystallite size and non-uniform strain.

Translating microcalcification biomarker information into the laboratory: A preliminary assessment utilizing core biopsies obtained from sites of mammographic calcification.

Rationale and objectives: The potential of breast microcalcification chemistry to provide clinically valuable intelligence is being increasingly studied. However, acquisition of crystallographic details has, to date, been limited to high brightness, synchrotron radiation sources. This study, for the first time, evaluates a laboratory-based system that interrogates histological sections containing microcalcifications. The principal objective was to determine the measurement precision of the laboratory system and assess whether this was sufficient to provide potentially clinical valuable information. Materials and methods: Sections from 5 histological specimens from breast core biopsies obtained to evaluate mammographic calcification were examined using a synchrotron source and a laboratory-based instrument. The samples were chosen to represent a significant proportion of the known breast tissue, mineralogical landscape. Data were subsequently analysed using conventional methods and microcalcification characteristics such as crystallographic phase, chemical deviation from ideal stoichiometry and microstructure were determined. Results: The crystallographic phase of each microcalcification (e.g., hydroxyapatite, whitlockite) was easily determined from the laboratory derived data even when a mixed phase was apparent. Lattice parameter values from the laboratory experiments agreed well with the corresponding synchrotron values and, critically, were determined to precisions that were significantly greater than required for potential clinical exploitation. Conclusion: It has been shown that crystallographic characteristics of microcalcifications can be determined in the laboratory with sufficient precision to have potential clinical value. The work will thus enable exploitation acceleration of these latent microcalcification features as current dependence upon access to limited synchrotron resources is minimized.

Conical shell X-ray beam tomosynthesis and micro-computed tomography for microarchitectural characterisation.

Bone quality is commonly used to diagnose bone diseases such as osteoporosis, with many studies focusing on microarchitecture for fracture prediction. In this study a bovine distal femur was imaged using both micro-computed tomography (µCT) and tomosynthesis using focal construct geometry (FCG) for comparison of microarchitectural parameters. Six regions of interest (ROIs) were compared between the two imaging modalities, with both global and adaptive methods used to binarize the images. FCG images were downsampled to the same pixel size as the µCT images. Bone morphometrics were determined using BoneJ, for each imaging modality, binarization technique and ROI. Bone area/total area was found to have few significant differences between FCG and µCT (p < 0.05 for two of six ROIs). Fractal Dimension had only one significant difference (p < 0.05 for one of six ROIs) between µCT and downsampled FCG (where pixel size was equalized). Trabecular thickness and trabecular spacing were observed to follow trends as observed for the corresponding µCT images, although many absolute values were significantly different (p < 0.05 for between one and six ROIs depending on image types used). This study demonstrates the utility of tomosynthesis for measurement of microarchitectural morphometrics.

A pilot study: effect of irisin on trabecular bone in a streptozotocin-induced animal model of type 1 diabetic osteopathy utilizing a micro-CT.

Background: Osteoporosis is a significant co-morbidity of type 1 diabetes mellitus (DM1) leading to increased fracture risk. Exercise-induced hormone 'irisin' in low dosage has been shown to have a beneficial effect on bone metabolism by increasing osteoblast differentiation and reducing osteoclast maturation, and inhibiting apoptosis and inflammation. We investigated the role of irisin in treating diabetic osteopathy by observing its effect on trabecular bone. Methods: DM1 was induced by intraperitoneal injection of streptozotocin 60 mg/kg body weight. Irisin in low dosage (5 µg twice a week for 6 weeks I/P) was injected into half of the control and 4-week diabetic male Wistar rats. Animals were sacrificed six months after induction of diabetes. The trabecular bone in the femoral head and neck was analyzed using a micro-CT technique. Bone turnover markers were measured using ELISA, Western blot, and RT-PCR techniques. Results: It was found that DM1 deteriorates the trabecular bone microstructure by increasing trabecular separation (Tb-Sp) and decreasing trabecular thickness (Tb-Th), bone volume fraction (BV/TV), and bone mineral density (BMD). Irisin treatment positively affects bone quality by increasing trabecular number p < 0.05 and improves the BMD, Tb-Sp, and BV/TV by 21-28%. The deterioration in bone microarchitecture is mainly attributed to decreased bone formation observed as low osteocalcin and high sclerostin levels in diabetic bone samples p < 0.001. The irisin treatment significantly suppressed the serum and bone sclerostin levels p < 0.001, increased the serum CTX1 levels p < 0.05, and also showed non-significant improvement in osteocalcin levels. Conclusions: This is the first pilot study to our knowledge that shows that a low dose of irisin marginally improves the trabecular bone in DM1 and is an effective peptide in reducing sclerostin levels.

Exploration of utility of combined optical photothermal infrared and Raman imaging for investigating the chemical composition of microcalcifications in breast cancer.

Microcalcifications play an important role in cancer detection. They are evaluated by their radiological and histological characteristics but it is challenging to find a link between their morphology, their composition and the nature of a specific type of breast lesion. Whilst there are some mammographic features that are either typically benign or typically malignant often the appearances are indeterminate. Here, we explore a large range of vibrational spectroscopic and multiphoton imaging techniques in order to gain more information about the composition of the microcalcifications. For the first time, we validated the presence of carbonate ions in the microcalcifications by O-PTIR and Raman spectroscopy at the same time, the same location and the same high resolution (0.5 µm). Furthermore, the use of multiphoton imaging allowed us to create stimulated Raman histology (SRH) images which mimic histological images with all chemical information. In conclusion, we established a protocol for efficiently analysing the microcalcifications by iteratively refining the area of interest.

Microcalcification crystallography as a potential marker of DCIS recurrence.

Ductal carcinoma in-situ (DCIS) accounts for 20-25% of all new breast cancer diagnoses. DCIS has an uncertain risk of progression to invasive breast cancer and a lack of predictive biomarkers may result in relatively high levels (~ 75%) of overtreatment. To identify unique prognostic biomarkers of invasive progression, crystallographic and chemical features of DCIS microcalcifications have been explored. Samples from patients with at least 5-years of follow up and no known recurrence (174 calcifications in 67 patients) or ipsilateral invasive breast cancer recurrence (179 microcalcifications in 57 patients) were studied. Significant differences were noted between the two groups including whitlockite relative mass, hydroxyapatite and whitlockite crystal maturity and, elementally, sodium to calcium ion ratio. A preliminary predictive model for DCIS to invasive cancer progression was developed from these parameters with an AUC of 0.797. These results provide insights into the differing DCIS tissue microenvironments, and how these impact microcalcification formation.

Investigating pair distribution function use in analysis of nanocrystalline hydroxyapatite and carbonate-substituted hydroxyapatite.

Hydroxyapatite (HA) is a complex material, which is often nanocrystalline when found within a biological setting. This work has directly compared the structural characteristics derived from data collected using a conventional laboratory-based X-ray diffractometer with those collected from a dedicated pair distribution function (PDF) beamline at Diamond Light Source. In particular, the application of PDF analysis methods to carbonated HA is evaluated. 20 synthetic samples were measured using both X-ray diffraction (XRD) and PDFs. Both Rietveld refinement (of laboratory XRD data) and real-space refinement (of PDF data) were used to analyse all samples. The results of Rietveld and real-space refinements were compared to evaluate their application to crystalline and nanocrystalline hydroxyapatite. Significant relationships were observed between real-space refinement parameters and increasing carbonate substitution. Understanding the local order of synthetic hydroxyapatite can benefit several fields, including both biomedical and clinical settings.

Breast calcification micromorphology classification.

OBJECTIVES: The importance of consistent terminology in describing the appearance of breast calcifications in mammography is well recognised. Imaging of calcifications using electron microscopy is a globally growing field of research. We therefore suggest that the time is ripe to develop a lexicon of terms for classifying the micromorphology of breast calcifications. METHODS: Calcifications within a wide range of histological sections of breast tissue, both benign and malignant, were imaged by Scanning Electron Microscopy (SEM). These images were examined, and the micromorphology of calcifications present was grouped to create a classification system. RESULTS: Based on the appearance of the calcifications observed, we propose five main categories for classification of the micromorphology of breast calcifications, namely, Dense Homogenous, Punctulate, Banded, Spongy and Aggregate. CONCLUSIONS: Use of the descriptive categories outlined here will help to ensure consistency and comparability of published observations on the micromorphology of breast calcifications. ADVANCES IN KNOWLEDGE: This is the first time a lexicon and classification system has been proposed for the micromorphology of breast calcifications, as observed by scanning electron microscopy of histological sections. This will facilitate comparability of observed relationships between micromorphology, mammographic appearance, chemistry and pathology.

New insights into the application of pair distribution function studies to biogenic and synthetic hydroxyapatites.

Biogenic and synthetic hydroxyapatites are confounding materials whose properties remain uncertain, even after years of study. Pair distribution function (PDF) analysis was applied to hydroxyapatites in the 1970's and 1980's, but this area of research has not taken full advantage of the relatively recent advances in synchrotron facilities. Here, synchrotron X-ray PDF analysis is compared to techniques commonly used to characterise hydroxyapatite (such as wide angle X-ray scattering, Fourier-transform infrared spectroscopy and thermogravimetric analysis) for a range of biogenic and synthetic hydroxyapatites with a wide range of carbonate substitution. Contributions to the pair distribution function from collagen, carbonate and finite crystallite size were examined through principal component analysis and comparison of PDFs. Noticeable contributions from collagen were observed in biogenic PDFs when compared to synthetic PDFs (namely r < 15 Å), consistent with simulated PDFs of collagen structures. Additionally, changes in local structure were observed for PDFs of synthetic hydroxyapatites with differing carbonate content, notably in features near 4 Å, 8 Å and 19 Å. Regression models were generated to predict carbonate substitution from peak position within the PDFs.

The use of µCT and fractal dimension for fracture prediction in osteoporotic individuals.

Osteoporosis (OP) is a widespread condition with commonly associated fracture sites at the hip, vertebra and wrist. This study examines the effects of age and osteoporosis on bone quality by comparing the efficacy of using parameters which indicate bone quality (both traditional clinical parameters such as bone mineral density (BMD), as well as apparent Young's modulus determined by finite element analysis, among others) to predict fracture. Non-fracture samples were collected from the femoral heads of 83 donors (44 males, 39 females), and fracture samples were obtained from the femoral heads of 17 donors (female). Microarchitectural parameters (Bone Volume/Total Volume [BV/TV], Bone Surface/Bone Volume [BS/BV], Tissue Mineral Density [TMD, etc.]) were measured from µCT of each sample as well as 2D and 3D fractal dimension (D2D and D3D respectively). A cube was cropped from µCT images and an isotropic hexahedral element was assigned to each voxel. Finite element analysis was used to calculate the Young's modulus for each sample. Overall, values for microarchitectural characteristics, fractal dimension measurements and Young's Modulus were consistent with values within literature. Significant correlations are observed between age and BV/TV for non-fracture males and females, as well as between age and volumetric BMD (vBMD) for the same groups. Significant differences are present between age-matched non-fracture and fracture females for BV/TV, BS/BV, vBMD, TMD, D2D, D3D, (p < 0.01 for all). Properties which are not age dependent are significantly different between age-matched non-fracture and fracture specimens, indicating OP is a disease, and not just an accelerated aging process.

Age-Related Trends in the Trabecular Micro-Architecture of the Medial Clavicle: Is It of Use in Forensic Science?

The mechanical and structural properties of bone are known to change significantly with age. Within forensic and archaeological investigations, the medial end of the clavicle is typically used for estimating the age-at-death of an unknown individual. Although, this region of the skeleton is of interest to forensic and clinical domains, alterations beyond the macro-scale have not been fully explored. For this study, non-destructive micro-computed tomography (µ-CT) was employed to characterize structural alterations to the cancellous bone of the medial clavicle. Fresh human cadaveric specimens (12-59 years) obtained at autopsy were utilized for this study, and were scanned with a voxel size of ~83 µm. Morphometric properties were quantified and indicated that the bone volume, connectivity density, mineral density, and number of trabeculae decreased with age, while the spacing between the trabeculae increased with age. In contrast to other sub-regions of the skeleton, trabecular thickness, and degree of anisotropy did not correlate with age. Collectively, this could suggest that the network is becoming increasingly perforated with age rather than exhibiting trabecular thinning. These results are used in the context of deriving a potential protocol for forensic investigations by using this particular and largely unexplored region of the skeleton, and provide inspiration for future experiments concerning micro-architectural and small scale changes in other regions of the human skeleton.

Age-Related Changes in Femoral Head Trabecular Microarchitecture.

Osteoporosis is a prevalent bone condition, characterised by low bone mineral density and increased fracture risk. Currently, the gold standard for identifying osteoporosis and increased fracture risk is through quantification of bone mineral density using dual energy X-ray absorption. However, many studies have shown that bone strength, and consequently the probability of fracture, is a combination of both bone mass and bone 'quality' (architecture and material chemistry). Although the microarchitecture of both non-fracture and osteoporotic bone has been previously investigated, many of the osteoporotic studies are constrained by factors such as limited sample number, use of ovariectomised animal models, and lack of male and female discrimination. This study reports significant differences in bone quality with respect to the microarchitecture between fractured and non-fractured human femur specimens. Micro-computed tomography was utilised to investigate the microarchitecture of femoral head trabecular bone from a relatively large cohort of non-fracture and fracture human donors. Various microarchitectural parameters have been determined for both groups, providing an understanding of the differences between fracture and non -fracture material. The microarchitecture of non-fracture and fracture bone tissue is shown to be significantly different for many parameters. Differences between sexes also exist, suggesting differences in remodelling between males and females in the fracture group. The results from this study will, in the future, be applied to develop a fracture model which encompasses bone density, architecture and material chemical properties for both female and male tissues.