The use of laser spectroscopy to investigate bone disease in King Henry VIII's sailors

The Mary Rose was King Henry VIII's flagship before it sank in battle on the 19th July 1545. Over four hundred men went down with the ship and the environment of the Solent meant their remains were quickly covered in silt. Between 1979 and 1982 the remains of 179 individuals were recovered and examined as part of the excavation of the Mary Rose. The anaerobic environment created by the silt preserved the sailors' bones in remarkable condition and to date much has been learnt about life on the ship. In this study we used Raman spectroscopy (a non-destructive technique), to investigate the chemistry of the human bones, specifically for the identification of disease in archaeological specimens, for the first time. Raman data were collected from five anatomically normal tibiae and five tibiae that were bowed (individuals suspected to have suffered from bone disease in childhood). The data were processed using multivariate analysis (principal component analysis) and results showed the presence of chemical abnormalities in the bowed bones which resulted in the separation of the bones into two clearly defined groups, normal and bowed

Is the collagen primed for mineralization in specific regions of the Turkey tendon?:an investigation of the protein-mineral interface using Raman spectroscopy

The tendons in the turkey leg have specific well-defined areas which become mineralized as the animal ages and they are a thoroughly characterized model system for studying the mineralization process of bone. In this study, nondestructive Raman spectroscopic analysis was used to explore the hypothesis that regions of the turkey tendon that are associated with mineralization exhibit distinct and observable chemical modifications of the collagen prior to the onset of mineralization. The Raman spectroscopy features associated with mineralization were identified by probing (on the micrometer scale) the transition zone between mineralized and nonmineralized regions of turkey leg tendons. These features were then measured in whole tendons and identified in regions of tendon which are destined to become rapidly mineralized around 14 weeks of age. The data show there is a site-specific difference in collagen prior to the deposition of mineral, specifically the amide III band at 1270 cm(-1) increases as the collagen becomes more ordered (increased amide III:amide I ratio) in regions that become mineralized compared to collagen destined to remain nonmineralized. If this mechanism were present in materials of different mineral fraction (and thus material properties), it could provide a target for controlling mineralization in metabolic bone disease

Measurement of abnormal bone composition in vivo using noninvasive Raman spectroscopy

X-ray-based diagnostic techniques, which are by far the most widely used for diagnosing bone disorders and diseases, are largely blind to the protein component of bone. Bone proteins are important because they determine certain mechanical properties of bone and changes in the proteins have been associated with a number of bone diseases. Spatially Offset Raman Spectroscopy (SORS) is a chemically specific analytical technique that can be used to retrieve information noninvasively from both the mineral and protein phases of the bone material in vivo. Here we demonstrate that SORS can be used to detect a known compositional abnormality in the bones of a patient suffering from the genetic bone disorder, osteogenesis imperfecta, a condition which affects collagen. The confirmation of the principle that bone diseases in living patients can be detected noninvasively using SORS points the way to larger studies that focus on osteoporosis and other chronic debilitating bone diseases with large socioeconomic burdens

Discrimination of ivory from extant and extinct elephant species using Raman spectroscopy : A potential non-destructive technique for combating illegal wildlife trade

The use of elephant ivory as a commodity is a factor in declining elephant populations. Despite recent worldwide elephant ivory trade bans, mammoth ivory trade remains unregulated. This complicates law enforcement efforts, as distinguishing between ivory from extant and extinct species requires costly, destructive and time consuming methods. Elephant and mammoth ivory mainly consists of dentine, a mineralized connective tissue that contains an organic collagenous component and an inorganic component of calcium phosphate minerals, similar in structure to hydroxyapatite crystals. Raman spectroscopy is a non-invasive laser-based technique that has previously been used for the study of bone and mineral chemistry. Ivory and bone have similar biochemical properties, making Raman spectroscopy a promising method for species identification based on ivory. This study aimed to test the hypothesis that it is possible to identify differences in the chemistry of mammoth and elephant ivory using Raman spectroscopy. Mammoth and elephant tusks were obtained from the Natural History Museum in London, UK. Included in this study were eight samples of ivory from Mammuthus primigenius, two samples of carved ivory bangles from Africa (Loxodonta species), and one cross section of a tusk from Elephas maximus. The ivory was scanned using an inVia Raman micro spectrometer equipped with a x50 objective lens and a 785nm laser. Spectra were acquired using line maps and individual spectral points were acquired randomly or at points of interest on all samples. The data was then analysed using principal component analysis (PCA) with use of an in-house MATLAB script. Univariate analysis of peak intensity ratios of phosphate to amide I and III peaks, and carbonate to phosphate peaks showed statistical differences (

Electron Beam-Treated Enzymatically Mineralized Gelatin Hydrogels for Bone Tissue Engineering

Biological hydrogels are highly promising materials for bone tissue engineering (BTE) due to their high biocompatibility and biomimetic characteristics. However, for advanced and customized BTE, precise tools for material stabilization and tuning material properties are desired while optimal mineralisation must be ensured. Therefore, reagent-free crosslinking techniques such as high energy electron beam treatment promise effective material modifications without formation of cytotoxic by-products. In the case of the hydrogel gelatin, electron beam crosslinking further induces thermal stability enabling biomedical application at physiological temperatures. In the case of enzymatic mineralisation, induced by Alkaline Phosphatase (ALP) and mediated by Calcium Glycerophosphate (CaGP), it is necessary to investigate if electron beam treatment before mineralisation has an influence on the enzymatic activity and thus affects the mineralisation process. The presented study investigates electron beam-treated gelatin hydrogels with previously incorporated ALP and successive mineralisation via incubation in a medium containing CaGP. It could be shown that electron beam treatment optimally maintains enzymatic activity of ALP which allows mineralisation. Furthermore, the precise tuning of material properties such as increasing compressive modulus is possible. This study characterizes the mineralised hydrogels in terms of mineral formation and demonstrates the formation of CaP in dependence of ALP concentration and electron dose. Furthermore, investigations of uniaxial compression stability indicate increased compression moduli for mineralised electron beam-treated gelatin hydrogels. In summary, electron beam-treated mineralized gelatin hydrogels reveal good cytocompatibility for MG-63 osteoblast like cells indicating a high potential for BTE applications

Gold nanoparticles as a substrate in bio-analytical near-infrared surface-enhanced Raman spectroscopy

As biospectroscopy techniques continue to be developed for screening or diagnosis within a point-ofcare setting, an important development for this field will be high-throughput optimization. For many of these techniques, it is therefore necessary to adapt and develop parameters to generate a robust yet simple approach delivering high-quality spectra from biological samples. Specifically, this is important for surface-enhanced Raman spectroscopy (SERS) wherein there are multiple variables that can be optimised to achieve an enhancement of the Raman signal from a sample. One hypothesis is that "large" diameter (>100 nm) gold nanoparticles provide a greater enhancement at near-infrared (NIR) and infrared (IR) wavelengths than thos

Modification of heat-induced whey protein isolate hydrogel with highly bioactive glass particles results in promising biomaterial for bone tissue engineering

This study deals with the design and comprehensive evaluation of novel hydrogels based on whey protein isolate (WPI) for tissue regeneration. So far, WPI has been considered mainly as a food industry by-product and there are very few reports on the application of WPI in tissue engineering (TE). In this work, WPI-based hydrogels were modified with bioactive glass (BG), which is commonly used as a bone substitute material. Ready-to-use, sterile hydrogels were produced by a simple technique, namely heat-induced gelation. Two different concentrations (10 and 20% w/w) of sol–gel-derived BG particles of two different sizes (2.5 and <45 µm) were compared. µCT analysis showed that hydrogels were highly porous with almost 100% pore interconnectivity. BG particles were generally homogenously distributed in the hydrogel matrix, affecting pore size, and reducing material porosity. Thermal analysis showed that the presence of BG particles in WPI matrix reduced water content in hydrogels and improved their thermal stability. BG particles decreased enzymatic degradation of the materials. The materials underwent mineralization in simulated biological fluids (PBS and SBF) and possessed high radical scavenging capacity. In vitro tests indicated that hydrogels were cytocompatible and supported MG-63 osteoblastic cell functions

Vibrational spectroscopy and saliva- a rapid and non-invasive diagnostic tools for diabetes and oral cancer

Oral cancer is a complex malignant disease of the head and neck that results from uncontrolled cell multiplication. Diabetes, alternatively, is a chronic metabolic condition characterized by elevated glucose concentration. Studies suggest that diabetes is a risk factor for oral cancer, possibly due to the overproduction of reactive oxygen species and high levels of insulin-like growth factors. Current diagnostic methods for oral cancer are time-consuming, invasive, and subject to inter-observer variability, making novel approaches necessary to identify the disease and malignancies. Fourier-transform Infrared (FTIR) spectroscopy is a sensitive and reproducible analytical technique that detects vibrational modes of molecular bonds. This study aimed to use FTIR spectroscopy to identify early biomarkers of oral cancer in diabetic patients. The study used 10 control (healthy) samples and 10 oral cancerous (diabetic) samples, with 10 independent spectra acquired from each sample to detct intra-sample heterogeneity. Spectral biomarkers that contributed to spectral variation were identified in the regions of amide I and amide III. Four machine learning models were generated, with model 1 covering the range of 600-4000cm-1, model 2 covering 1000-1800cm-1, model 3 covering 1000-1500cm-1, and model 4 covering 1800-3000cm-1, respectively. The data was analyzed by Principal component analysis (PCA), Linear discriminant analysis (LDA), k-nearest neighbors algorithm (k-NN), and support vector machine (SVM). The highest classifier, with a prediction accuracy of over 80%, was produced by applying SVM. The results support the use of FTIR spectroscopy with machine learning as an adjunct method to the gold standard in the diagnosis of oral cancer. We continued the project with saliva samples as a case-control study, where saliva samples (80) were collected from type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), and healthy controls (CON) were analyzed to identify specific molecular spectral signatures. Spectral analysis revealed unique vibrational modes in diabetic saliva samples compared to non-diabetic samples. Spectral biomarkers in the regions of Amide I and carbohydrates were discovered, along with subtle differences in spectra during spectral and multivariate analysis that could provide a robust and novel non-invasive monitoring tool for diabetes. FTIR imaging has advantages over conventional methods, as it is a label-free, non-disruptive technique requiring only a small amount of sample. It provides a biochemical fingerprinting that includes information about the structure, content, and chemical modification of any major biomolecules in the tested sample. In saliva samples, FTIR imaging provides information about modifications in proteins, DNA/RNA, and carbohydrates induced by external interventions. The spectral salivary biomarkers discovered using univariate and multivariate analysis may provide a novel and robust alternative for diabetes monitoring using non-invasive technology