Physiochemical modifications to bone mineral

Bone is a complex composite material consisting of three main components: a mineral phase structurally similar to calcium hydroxyapatite (HAp), an organic matrix containing collagenous and non-collagenous proteins and, water. The complexity of bone has led to an abundance of literature across a wide range of disciplines, which have endeavoured to provide a greater understanding of this material. In particular, heated bone studies are prevalent in biomedicine where heat treatment is often used to sterilise bone material required for xeno– and allo- grafts, in forensic science where species differentiation of unknown heated bone specimens would prove invaluable and in archaeology, where heated bone material often provides information about the cooking and funeral practices of our ancestors. Unfortunately, many of these studies are largely observational and some of the processes and mechanisms associated with heated bone are largely assumed and in some instances ambiguous. Over 1000 biological and synthetic HAp specimens were utilised during this research to investigate the fundamental processes and mechanisms associated with unheated and heated bone. In particular, three controversial areas of bone research were considered: - in vivo HAp crystal size control, the relationship between the organic and mineral components of bone during heat treatment and the confounding effects of cooling on bone mineral during heat treatment. This was achieved by considering the chemical composition of unheated biological and synthetic HAp specimens, and heated bone specimens from various species including human. The results of this thesis demonstrate that an intrinsic rather than extrinsic source may be responsible for in vivo biological HAp crystal size control, a concept which has not previously be considered. The results have also shown bone mineral crystallisation during heat treatment is promoted by the organic matrix and, cooling has an impact on both crystallisation and thermal decomposition of HAp during heat treatment. This research has also questioned the use of current X-ray diffraction (XRD) refinement techniques with nanocrystalline materials such as bone, to determine crystalline size and strain. Further interpretation of the results questioned whether heated bone data is comparable between research groups, whether it was possible to create a time and temperature predictive model for heated bone and whether human bone is statistically different from other bone types when dynamically heated. Due to the fundamental nature of this research, it is expected the results will have an impact across a wide range of disciplines including biomedicine, forensic science and archaeology

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

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 ∼83µm. 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

Fracture toughness of the cancellous bone of FNF femoral heads in relation to its microarchitecture

This study considers the relationship between microarchitecture and mechanical properties for cancellous bone specimens collected from a cohort of patients who had suffered fractured necks of femur. OP is an acute skeletal condition with huge socioeconomic impact [1] and it is associated with changes in both bone quantity and quality [2], which affect greatly the strength and toughness of the tissue [3].Support was provided by the EPSRC (EP/K020196: Point-ofCare High Accuracy Fracture Risk Prediction), the UK Department of Transport under the BOSCOS (Bone Scanning for Occupant Safety) project, and approved by Gloucester and Cheltenham NHS Trust hospitals under ethical consent (BOSCOS – Mr. Curwen CI REC ref 01/179G)

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

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

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) 41 leading to increased fracture risk. Exercise-induced hormone 'irisin' in low dosage has been 42 shown to have a beneficial effect on bone metabolism by increasing osteoblast differentiation 43 and reducing osteoclast maturation, and inhibiting apoptosis and inflammation. We investigated 44 the role of irisin in treating diabetic osteopathy by observing its effect on trabecular bone. 45 Methods. DM1 was induced by intraperitoneal injection of streptozotocin 60 mg/kg body 46 weight. Irisin in low dosage (5 μg twice a week for 6 weeks I/P) was injected into half of the 47 control and 4-week diabetic male Wistar rats. Animals were sacrificed six months after induction 48 of diabetes. The trabecular bone in the femoral head and neck was analyzed using a micro-CT 49 technique. Bone turnover markers were measured using ELISA, Western blot, and RT-PCR 50 techniques. 51 Results. It was found that DM1 deteriorates the trabecular bone microstructure by increasing 52 trabecular separation (Tb-Sp) and decreasing trabecular thickness (Tb-Th), bone volume fraction 53 (BV/TV), and bone mineral density (BMD). Irisin treatment positively affects bone quality by 54 increasing trabecular number p < 0.05 and improves the BMD, Tb-Sp, and BV/TV by 21-28%. 55 The deterioration in bone microarchitecture is mainly attributed to decreased bone formation 56 observed as low osteocalcin and high sclerostin levels in diabetic bone samples p < 0.001. The 57 irisin treatment significantly suppressed the serum and bone sclerostin levels p < 0.001, 58 increased the serum CTX1 levels p < 0.05, and also showed non-significant improvement in 59 osteocalcin levels. 60 Conclusions. This is the first pilot study to our knowledge that shows that a low dose of irisin 61 marginally improves the trabecular bone in DM1 and is an effective peptide in reducing 62 sclerostin levels

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.Engineering and Physical Sciences Research Council (EPSRC): EP/T034238/1. Royal Society Wolfson Fellowship: RSWF/R1/180012. Diamond Light Source, instrument I15-1: proposal ee18638

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