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The occurrence of spontaneous humeral fractures in primiparous dairy cows from New Zealand prompted the study of bone material from affected cows to further characterize this condition and to outline a likely pathogenesis. Previous studies indicate that these cows developed osteoporosis due to periods of suboptimal bone formation followed by increased bone resorption during the period of lactation complicated by copper deficiency. We hypothesized that there are significant differences in the chemical composition/bone quality in bones from cows with spontaneous humeral fracture compared to cows without humeral fractures. In this study, Raman and Fourier transform infrared spectroscopy band ratios were, for the first time, measured, calculated, and compared in bone samples from 67 primiparous dairy cows that suffered a spontaneous fracture of the humerus and 14 age-matched post-calving cows without humeral fractures. Affected bone showed a significantly reduced mineral/matrix ratio, increased bone remodeling, newer bone tissue with lower mineralization and, lower carbonate substitution, and reduced crystallinity. As such, is likely that these have detrimentally impacted bone quality and strength in affected cows.
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Bioprinting is an emerging technology involved in the fabrication of three-dimensional tissue constructs for the repair and regeneration of various tissues and organs. Collagen, a natural protein found abundantly in the extracellular matrix of several tissues, can be extracted from collagen-rich tissues of animals such as sheep, cows, rats, pigs, horses, birds, and marine animals. However, due to the poor printability of collagen bioinks, biocompatible collagen scaffolds that mimic the extracellular matrix (ECM) are difficult to fabricate using bioprinting techniques. Gelatin methacrylate (GelMA), a semi-synthetic polymer with tunable physical and chemical properties, has been found to be a promising biomaterial in various bioprinting applications. The printability of collagen can be improved by combining it with semi-synthetic polymers such as GelMA to develop hybrid hydrogels. Such hybrid hydrogels printed have also been identified to have enhanced mechanical properties. Hybrid GelMA meshes have not previously been prepared with collagen from ovine sources. This study provides a novel comparison between the properties of hybrid meshes with ovine skin and bovine hide collagen. GelMA (8% w/v) was integrated with three different concentrations (0.5%, 1%, and 2%) of bovine and ovine collagen forming hybrid hydrogels inks that were printed into meshes with enhanced properties. The maximum percentage of collagen suitable for integration with GelMA, forming hybrid hydrogels with a stable degradation rate was 1%. The water-soluble nature of ovine collagen promoted faster degradation of the hybrid meshes, although the structural crosslinking was identified to be higher than bovine hybrid meshes. The 1% bovine collagen hybrid meshes stood out in terms of their stable degradation rates.
RESUMEN
Numerous cases of spontaneous humeral fracture in primiparous dairy cows from New Zealand have prompted the study of the condition to establish probable causes or risk factors associated with the condition. Previous studies identified inadequate protein-calorie malnutrition as an important contributory factor. Earlier case studies also reported that ~50% of cows have low liver and/or serum copper concentration at the time of humeral fracture. Because copper is so closely associated with the formation of collagen cross-links, the aim of this study was to compare collagen and collagen crosslink content in the humerus from primiparous cows with and without humeral fractures and to determine the role of copper in the occurrence of these fractures. Humeri were collected from cows with and without humeral fractures, ground, and the collagen and collagen cross-link content measured using high-performance liquid chromatography. Collagen content was significantly higher in the humeri of cows without humeral fractures, while total collagen crosslink content was significantly higher in the humerus of cows with humeral fractures. These results indicate other factor/s (e.g., protein-calorie undernutrition) might be more important than the copper status in the occurrence of humeral fractures in dairy cows in New Zealand.
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Carotenoids are powerful antioxidants capable of helping to protect the skin from the damaging effects of exposure to sun by reducing the free radicals in skin produced by exposure to ultraviolet radiation, and they may also have a physical protective effect in human skin. Since carotenoids are lipophilic molecules which can be ingested with the diet, they can accumulate in significant quantities in the skin. Several studies on humans have been conducted to evaluate the protective function of carotenoids against various diseases, but there is very limited published information available to understand the mechanism of carotenoid bioavailability in animals. The current study was conducted to investigate the skin carotenoid level (SCL) in two cattle skin sets - weaners with an unknown feeding regime and New Generation Beef (NGB) cattle with monitored feed at three different ages. Rapid analytical and sensitive Raman spectroscopy has been shown to be of interest as a powerful technique for the detection of carotenoids in cattle skin due to the strong resonance enhancement with 532 nm laser excitation. The spectral difference of both types of skin were measured and quantified using univariate and linear discriminant analysis. SCL was higher in NGB cattle than weaners and there is a perfect classification accuracy between weaners and NGB cattle skin using carotenoid markers as a basis. Further work carried out on carotenoid rich NGB cattle skin of 8, 12 and 24 months of age identified an increasing trend in SCL with age. The present work validated the ability of Raman spectroscopy to determine the skin carotenoid level in cattle by comparing it with established HPLC methods. There is an excellent correlation of R2 = 0.96 between the two methods that could serve as a model for future application for larger population studies.
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Collagen-based hydrogels are investigated extensively in tissue engineering for their tunable physiochemical properties, biocompatibility and biodegradability. However, the effect of the integrity of the collagen triple helical structure on biodegradability is yet to be studied. In this study, we monitored the degradation of intact collagen (C-coll) and hydrolyzed collagen (D-coll) hydrogels in collagenase Clostridium histolyticum to understand their degradation process. Our results show that when peptides are present on the surface of the fibrils of D-coll hydrogels, cleavage of amide bonds occur at a much higher rate. The fibrillar structure of D-coll hydrogel results in a more pronounced breakdown of the gel network and dissolution of collagen peptides. The results from this work will improve the understanding of enzymatic degradation and the resulting bioabsorption of collagen materials used in drug delivery systems and scaffolds.
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New Zealand deer farming centres on the production of meat and velvet antler. Velvet antler removal is a painful procedure and currently, New Zealand Animal Welfare regulations dictate surgical removal of velvet antlers under lignocaine anaesthesia. To improve our knowledge on the efficacy and duration of other local anaesthetics to mitigate pain after antler removal, it is important to accurately assess and quantify pain arising from antler removal. Therefore, the current study was designed to validate mechanical nociceptive threshold (MNT) testing using a Wagner hand-held algometer, and to apply this methodology to assess the efficacy and duration of action of articaine for antler removal in deer. Baseline force (N) required to elicit the nociceptive response was recorded in 40 yearling male red deer on three alternate days. Ten of the 40 animals were selected for antler removal after administration of 4% articaine hydrochloride as a ring block. The duration of analgesic efficacy of articaine was assessed by algometry across 5 time points. There was a significant difference in MNTs among the three days (day 3 versus day 1 (p < 0.0001), day 2 versus day 1 (p < 0.0001), and day 1 versus day 2 (p < 0.01)). Positive correlations were observed between weight, antler length and thresholds. The MNT values remained above 20N for 6 h after removal of velvet antlers under the articaine ring block. This study provides valuable information about the use of MNT in red deer. These findings lay a foundation for future studies in the topics of peri-operative and postoperative pain management in deer antler removal, and a possible alternative use for articaine.
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Collagen is an important biomacromolecule, making up the majority of the extracellular matrix in animal tissues. Naturally occurring crosslinks in collagen stabilize its intermolecular structure in vivo, whereas chemical treatments for introducing synthetic crosslinks are often carried out ex vivo to improve the physical properties or heat stability of the collagen fibres for applications in biomaterials or leather production. Effective protection of intrinsic natural crosslinks as well as allowing them to contribute to collagen stability together with synthetic crosslinks can reduce the need for chemical treatments. However, the contribution of these natural crosslinks to the heat stability of collagen fibres, especially in the presence of synthetic crosslinks, is as yet unknown. Using synchrotron small-angle X-ray scattering, the in situ role of natural and synthetic crosslinks on the stabilization of the intermolecular structure of collagen in skins was studied. The results showed that, although natural crosslinks affected the denaturation temperature of collagen, they were largely weakened when crosslinked using chromium sulfate. The development of synergistic crosslinking chemistries could help retain the intrinsic chemical and physical properties of collagen-based biological materials.
Asunto(s)
Colágeno/efectos de la radiación , Sincrotrones , Animales , Bovinos , Compuestos de Cromo/química , Reactivos de Enlaces Cruzados/química , Estructura Molecular , Dispersión del Ángulo Pequeño , Sulfatos/química , Rayos XRESUMEN
Carotenoids, powerful anti-oxidants, play a significant role in protecting the skin from oxidation and help in balancing the redox status of skin. This study was aimed at investigating cattle skin to identify carotenoids in the lower epidermis (grain) and dermis (corium) layers for classification using Raman spectroscopy which is a powerful technique for the detection of carotenoids in cattle skin due to the strong resonance enhancement with 532 nm laser excitation. The spectral differences identified between these two layers were quantified by the univariate analysis of Raman peak heights and partial least squares (PLS) analysis. We compared the performance of the Raman spectroscopy method with the standard method, high performance liquid chromatography. The univariate analysis results demonstrated that the lower epidermis of the skin has a higher concentration of carotenoid than dermis using the carotenoid Raman peaks at 1151 cm-1 and 1518 cm-1. The carotenoid Raman intensity was linearly correlated with the total carotenoid concentration determined by standard HPLC methods. Partial Least Squares Regression analysis gives excellent results with R 2 = 0.99. Our results indicate that Raman spectroscopy is a potential tool to determine carotenoids in cattle skin with high precision.
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Skin is made up of mainly collagen type I and its structure is stabilised by the formation of covalent immature and mature crosslinks. In this study, collagen immature crosslink hydroxylysinonorleucine (HLNL) was isolated from bovine skin in high purity using two sequential purification steps. These consisted of preparative fibrous cellulose and size exclusion chromatography. The purified crosslink was then analysed using tandem mass spectrometry and high-resolution nuclear magnetic resonance (NMR) spectroscopy. The mass of singly and doubly charged ions of HLNL was 292.1865 and 146.5970 m/z and their optimised fragmentation energy was 17 keV and 5 keV, respectively. The 13C NMR of HLNL showed a doubled-up peak at 67.84 and 67.91 ppm which corroborated a diastereomeric form of collagen immature crosslink HLNL and both are chiroptically indistinguishable. The chemical structure was fully resolved using 1H, 13C and DEPT-135 high-resolution NMR spectroscopy and compared with other previous studies. We also obtained for the first time the 2D NMR spectra COSY and HSQC of HLNL. We therefore suggested that collagen organization into specific fibrils' orientation may be affected by the different configuration of these diastereomers of HLNL.
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Colágeno Tipo I/metabolismo , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/aislamiento & purificación , Dipéptidos/química , Dipéptidos/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Piel/metabolismo , Animales , Bovinos , Reactivos de Enlaces Cruzados/metabolismo , Dipéptidos/metabolismo , Imagen por Resonancia MagnéticaRESUMEN
Although the biomechanical properties of skin and its molecular components have been extensively studied, little research has been devoted to understanding the links between them. Here, a comprehensive analysis of the molecular components of deer and cow skins was undertaken in order to understand the basis of their physical properties. These skins were chosen because they are known to be strong yet supple, exhibiting properties that have been exploited by man for centuries. Firstly, the tensile strength, tear strength and denaturation temperature of deer and cow skins were measured. Secondly, the organisation of the collagen fibrils and presence of glycosaminoglycans in each skin was investigated using polarising microscopy (PM), laser scanning confocal microscopy (LSCM), transmission electron microscopy (TEM), nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS). Finally, amino acid, crosslink and glycosaminoglycan analyses were carried out on both skins in the study. The results of the study showed that individual physical properties such as tensile strength of the skin are derived from different combinations of biomolecular components which are reflected in collagen architecture. The "wavy" organisation of collagen fibres in deer skin was associated with a small fibril diameter, uniform glycosaminoglycan distribution and higher proportion of trivalent crosslinks. In contrast, the collagen fibrils in cow skin were large, contained a diverse glycosaminoglycan distribution and a higher proportion of tetravalent crosslinks, resulting in straight fibres. This study showed for the first time that the relationship between the structure of collagen in skin and its biomechanical functions is complex, arising from different architectural and molecular features including organisation of collagen fibres, diameters of collagen fibrils, distribution and amount of glycosaminoglycans and types and concentrations of crosslinks.
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Colágeno/metabolismo , Piel/metabolismo , Animales , Bovinos , Ciervos , Glicosaminoglicanos/metabolismo , Ensayo de Materiales , Temperatura , Resistencia a la TracciónRESUMEN
Pyridinoline and deoxypyridinoline crosslinks are biomarkers found in urine for collagen degradation in bone turnover. For the first time, a rapid, sensitive, and ion-pairing free method is described for the analysis of pyridinoline and deoxypyridinoline using ultra-high performance liquid chromatography with Cogent Diamond Hydride column and detection by Q Exactive hybrid quadrupole-orbitrap high resolution accurate mass spectrometry. The separation was achieved using both isocratic and gradient conditions and run time <5 min under isocratic conditions of 20% acetonitrile in water containing 0.1% formic acid. Pyridoxine was used as an internal standard and relative standard deviation of the retention times of both pyridinoline and deoxypyridinoline were <1%. The limit of detection was 0.082 ± 0.023 µM for pyridinoline and 0.118 ± 0.052 µM for deoxypyridinoline. The limit of quantitation was 0.245 ± 0.070 µM for pyridinoline and 0.354 ± 0.157 µM for deoxypyridinoline. The method was validated by the detection and quantitation of both pyridinoline and deoxypyridinoline in skin and urine samples.
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Aminoácidos/orina , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Piel/química , Aminoácidos/análisis , Animales , Bovinos , Cromatografía Líquida de Alta Presión/instrumentación , Perros , Femenino , Conejos , Silicatos/química , Orina/químicaRESUMEN
In this protocol we describe the separation of collagen crosslinks in biological tissues and samples including skin, tendon, cartilage, bone and urine. The existing methods use either cation exchange chromatography followed by post-column derivatization with ninhydrin or reverse phase chromatography with mass spectrometry detection. The cation exchange chromatography method has limited sensitivity and long run times while reverse phase chromatography requires strong ion-pairing. In this method, the sample containing crosslinks is applied on a diamond hydride column using water and acetonitrile solvents containing 0.1% (w/v) formic acid. Eight crosslinks are eluted separately from the column and detected by mass spectrometry in the sub-pmol range. By using this method, it is possible to separate all crosslinks of collagen in several biological samples without the need for ion-pairing agent or derivatization for detection.
RESUMEN
We have developed a novel chromatographic analytical method for the simultaneous quantitation of collagen crosslinks. Seven non-derivatised crosslinks could be separated on a Cogent Diamond Hydride HPLC column using either isocratic or gradient conditions then detected by mass spectrometry. The total run time was less than 10min which is significantly shorter than that previously reported. This is the first method in which histidinohydroxylysinonorleucine (HHL) and histidinohydroxymero-desmosine (HHMD) were separated and identified by mass spectrometry without the need for pre- or post-column derivatization. The CVs of the retention times of all seven crosslinks were less than 1% and the limit of detection (LOD) and the limits of quantitation (LOQ) were 0.07-0.13pmol/µL and 0.20-0.38pmol/µL, respectively. This novel method was used for the routine analysis and quantitation of crosslinks in different animal skins in which potential new collagen crosslinks were identified that are as yet undocumented.