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Intramuscular connective tissue (IMCT) is mainly composed of several fibrils (known as total collagen (TCol)) linked between each other by different chemical cross-links (CLs), the whole being embedded in a matrix of proteoglycans (PGs). In the field of beef quality, there is limited information on the role of CLs and PGs. Accordingly, several authors suggest that, to investigate the role of IMCT, it is important to investigate them just like TCol and insoluble collagen (ICol). In muscle, there are two other components, the muscle fibres and intramuscular fat (IMF) content. There are limited data on the relationships between these three components of muscle and then on possibility to independently manipulate these characteristics in order to control the final quality of meat. The present study aimed to investigate whether consistent relationships exist between these different components of muscle. Therefore, the present study compared four muscles of two cattle types (dairy and beef) to determine associations between TCol, ICol, CLs and PGs. Data were analysed across and within muscle (M) and animal type (AT) based on residuals. There was a strong M and AT effect for all muscle characteristics and an interaction M × AT for type I muscle fibres and IMF. Correlations between TCol, ICol and their CLs were M- and AT-independent. Total proteoglycans were positively correlated with TCol and ICol in a muscle-dependent manner irrespective of AT, but no correlation was found with CLs. On the contrary, CLs were negatively correlated with the ratio TPGs : TCol in an M-dependent manner, irrespective of AT. TCol, ICol and CLs were positively and negatively correlated with type IIA and IIB+X muscle fibres only in longissimus thoracis (LT) muscle, regardless the AT. Insoluble collagen was the only parameter of IMCT to be correlated with type I muscle fibres but only in LT muscle, irrespective of AT. There was no correlation between PGs and muscle fibre types, but PGs were the only IMCT component to be related with IMF in an M-dependent manner, irrespective of AT. Finally, there was no correlation between muscle fibre types and IMF content within M and AT. This study revealed that there is a strong relationship between IMCT components irrespective of M, an M-dependent relationship between the IMCT components and muscle fibre types and few (only with PGs) or no relationship between IMF and IMCT and muscle fibres.
In recent years, tissue engineering has helped to reduce hospital stays and deaths caused by skin wounds. Scaffolds are one of the main factors that influence the success of any tissue graft. Collagen is one of the main components of the extracellular matrix, and there has been much interest in new sources for application as a biomaterial. In this work, a tissue engineering scaffold was developed using the electrospinning technique. The chicken skin was used as an alternative source to obtain collagen. The combination of this collagen with elastin was successfully electrospun, and a distribution of diameters was obtained, less than 100 nm. In vitro tests showed the adhesion and proliferation of the cells, as well as an absence of cytotoxicity from non–cross-linked scaffolds and scaffolds that were cross-linked with carbonyldiimidazole. The structure and composition of the developed scaffolding provide a favorable environment for cell growth and generating a skin substitute.
Collagen microstructure is closely related to the mechanical properties of tissues and affects cell migration through the extracellular matrix. To study these structures, three-dimensional (3D) in vitro collagen-based gels are often used, attempting to mimic the natural environment of cells. Some key parameters of the microstructure of these gels are fiber orientation, fiber length, or pore size, which define the mechanical properties of the network and therefore condition cell behavior. In the present study, an automated tool to reconstruct 3D collagen networks is used to extract the aforementioned parameters of gels of different collagen concentration and determine how their microstructure is affected by the presence of cells. Two different experiments are presented to test the functionality of the method: first, collagen gels are embedded within a microfluidic device and collagen fibers are imaged by using confocal fluorescence microscopy; second, collagen gels are directly polymerized in a cell culture dish and collagen fibers are imaged by confocal reflection microscopy. Finally, we investigate and compare the collagen microstructure far from and in the vicinities of MDA-MB 23 cells, finding that cell activity during migration was able to strongly modify the orientation of the collagen fibers and the porosity-related values.
The consumption of high-Ca, high-protein dairy foods (i.e. milk, cheese, yogurt) is advocated for bone health across the lifespan to reduce the risk of low-trauma fractures. However, to date, the anti-fracture efficacy of dairy food consumption has not been demonstrated in randomised controlled trials but inferred from cross-sectional and prospective studies. The anti-fracture efficacy of dairy food consumption is plausible, but testing this requires a robust study design to ensure outcomes are suitably answering this important public health question. The evidence of skeletal benefits of dairy food consumption is equivocal, not because it may not be efficacious but because the study design and execution are often inadequate. The key issues are compliance with dietary intervention, dropouts, sample sizes and most importantly lack of deficiency before intervention. Without careful appraisal of the design and execution of available studies, precarious interpretations of outcomes may be made from these poorly designed or executed studies, without consideration of how study design may be improved. Dairy food interventions in children are further hampered by heterogeneity in growth: in particular sex and maturity-related differences in the magnitude, timing, location and surface-specific site of bone accrual. Outcomes of studies combining children of different sexes and maturity status may be masked or exaggerated by these differences in growth, so inaccurate conclusions are drawn from results. Until these critical issues in study design are considered in future dairy food interventions, the anti-fracture efficacy of dairy food consumption may remain unknown and continue to be based on conjecture.
Curcuma longa, also known as turmeric, has long been used as a medicinal herb with various biological effects. A hot water extract of C. longa (WEC) has been reported to show antioxidant and anti-inflammatory activity, but its effect on hepatic inflammation is poorly understood. In the present study, to investigate the effect of WEC on non-alcoholic steatohepatitis, C57BL/6J mice were fed a low-methionine, choline-deficient diet with 0·175 % WEC (WEC group) or without WEC (control group) for 6 or 12 weeks. Although hepatic steatosis was similar in the WEC group and the control group, WEC suppressed the elevation of plasma aspartate aminotransferase and alanine aminotransferase, which are markers of hepatocellular damage. Compared with the control group, the WEC group had higher hepatic levels of reduced glutathione and superoxide dismutase, as well as a lower hepatic level of thiobarbituric acid-reactive substances. WEC also reduced hepatic expression of mRNA for inflammatory factors, including TNF-α, IL-1β, IL-6, monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, F4/80 and CC motif chemokine receptor 2. Histological examination revealed that WEC suppressed hepatic recruitment of F4/80+ monocytes/macrophages and inhibited hepatic fibrosis. Furthermore, WEC inhibited hepatic expression of mRNA for molecules related to fibrosis, such as transforming growth factor-β, α-smooth muscle actin, type I collagen (α1-chain) and tissue inhibitor of matrix metalloproteinase-1. These findings suggest that dietary intake of WEC prevents the progression of non-alcoholic steatohepatitis by alleviating hepatic oxidative stress and inflammation.
The polyphenolic extract (PE) from extra virgin olive oil (EVOO) has been shown to possess important anti-inflammatory and joint protective properties in murine models of rheumatoid arthritis (RA). This study was designed to evaluate the effects of PE on IL-1β-activated human synovial fibroblasts SW982 cell line. PE from EVOO treatment inhibited IL-1β-induced matrix metalloproteases (P<0·001), TNF-α and IL-6 production (P<0·001). Similarly, IL-1β-induced cyclo-oxygenase-2 and microsomal PGE synthase-1 up-regulations were down-regulated by PE (P<0·001). Moreover, IL-1β-induced mitogen-activated protein kinase (MAPK) phosphorylation and NF-κB activation were ameliorated by PE (P<0·001). These results suggest that PE from EVOO reduces the production of proinflammatory mediators in human synovial fibroblasts; particularly, these protective effects could be related to the inhibition of MAPK and NF-κB signalling pathways. Taken together, PE from EVOO probably could provide an attractive complement in management of diseases associated with over-activation of synovial fibroblasts, such as RA.
Both genetic selection and increasing nutrient density for improving growth performance had inadvertently increased leg problems of meat ducks, which adversely affects animal welfare. We hypothesised that slowing weight gain with improving tibia quality probably enhanced tibial mechanical properties and alleviated leg deformities. Therefore, the present study aimed to evaluate the effect of graded Ca supplementation in a low-nutrient density (LND) diet on tibia composition and bone turnover in meat ducks. A total of 720 15-d-old male meat ducks were randomly assigned and fed a standard nutrient density positive control (PC) diet containing 0·9 % Ca, and four LND diets with 0·5, 0·7, 0·9 and 1·1 % Ca, respectively. Ducks fed the 0·5 % Ca LND diet and the PC diet had higher incidence of tibial dyschondroplasia (TD). When compared with the 0·5 % Ca LND diet, LND diets with ≥0·7 % Ca significantly improved tibia composition, microarchitecture and mechanical properties, and consequently decreased the incidence of TD. Furthermore, LND diets with ≥0·7 % Ca increased osteocyte-specific gene mRNA expression, blocked the expression of osteoblast differentiation marker genes including osteocalcin, collagenase-1 and alkaline phosphatase (ALP), and also decreased the expression of osteoclast differentiation genes, such as vacuolar-type H+-ATPase, cathepsin K and receptor activator of NF-κB. Meanwhile bone markers such as serum ALP, osteocalcin (both osteoblast markers) and tartrate-resistant acid phosphatase (an osteoclast marker) were significantly decreased in at least 0·7 % Ca treated groups. These findings indicated that LND diets with ≥0·7 % Ca decreased bone turnover, which subsequently increased tibia quality for 35-d-old meat ducks.
Tollund Man is one of the most famous Iron Age bog bodies due to his well-preserved head. Since he was unearthed in 1950 in Bjældskovdal, Denmark, he has been subjected to several scientific investigations, but until now no attempts to reconstruct his general diet through isotope analyses have been conducted. Furthermore, previous radiocarbon (14C) analyses have only been able to date him broadly to the 3rd–4th century BC. In this study, stable isotope measurements (δ13C, δ15N) on bone collagen from Tollund Man’s femur and rib showed that the diet of Tollund Man was terrestrial-based and that the crops he ate probably were grown on manured fields. Accelerator mass spectrometry (AMS) 14C dates were obtained on both the <30kDa and >30kDa fractions of ultrafiltered collagen. Results showed that the ultrafiltration removed contamination from older substances from the burial environment. The femur was dated to 2330±23 BP, the rib to 2322±30 BP. These dates statistically agree with a previously published AMS 14C age on skin. By combining the new dates with the previous date of his skin it was possible to narrow down the age of Tollund Man to the period 405–380 cal BC (95.4% confidence interval).
Human burials from the cemetery at the Rounala church, northern Sweden, were radiocarbon (14C) dated to shed light on the use of the cemetery. Carbon, nitrogen and sulfur stable isotope analysis of bone collagen from 19 distinct individuals indicated that these individuals had a mixed diet consisting of freshwater, marine and terrestrial resources. Dietary modeling using FRUITS was employed to calculate the contributions of the different resources for each individual. These data were then used to calculate individual ΔR values, taking into account freshwater and multiple marine reservoir effects, the latter caused by Baltic and Atlantic marine dietary inputs, respectively. 14C dating of tissues from modern freshwater fish species demonstrate a lack of a freshwater reservoir effect in the area. Two OxCal models were used to provide endpoint age estimates. The calibrated data suggest that the site’s cemetery was most likely in use already from the 14th century, and perhaps until at least the late 18th century.
Evaluation of eleven candidate probiotic Lactobacillus strains isolated from human milk showed that some of the strains were well endowed with desirable cell surface and attachment attributes. The cell surface properties (hydrophobicity, auto-aggregation, attachment to collagen and HT-29 monolayer) of probiotic Lactobacillus species of human milk origin were compared with reference probiotic/ non-probiotic species and pathogenic strains. The bacterial adhesion to hydrocarbons (BATH) was determined using three aliphatic (Chloroform, n-Hexane and n-Octane) and two aromatic (Toluene and Xylene) solvents. Maximum affinity of Lactobacillus strains towards chloroform and toluene indicated the presence of low electron acceptor/ acidic surface components on cell surface of most of the strains. The highest value of per cent hydrophobicity was recorded with chloroform in HM1 (L. casei) (97·10 ± 3·35%) and LGG (98·92 ± 1·24%). A moderate auto-aggregation attribute was observed in all of our Lactobacillus isolates. Only HM10, HM12 and HM13 exhibited comparatively enhanced precipitation rate after 7 h of incubation period. The adhesion potential to collagen matrix was highest in LGG (26·94 ± 5·83%), followed by HM1 (11·07 ± 3·54%) and HM9 (10·85 ± 1·74%) whereas, on HT-29 cells, HM8 (14·99 ± 3·61%), HM3 (13·73 ± 1·14%) and HM1 (11·21 ± 3·18%) could adhere effectively. In this manner, we noticed that although the cell surface properties and adhesion prospective of probiotic bacteria were strain dependent, five of our isolates viz. HM1, HM3, HM8, HM9 and HM10 exhibited promising cell surface properties, which could be further targeted as indigenous probiotic.
Higher δ15N values in bone collagen of mammoth (Mammuthus primigenius) compared with coeval large herbivores is a classic trait of the mammoth steppe. An exception applies to the Epigravettian site of Mezhyrich (ca. 18–17.4 ka cal BP) in the central East European plains, where mammoth bones have δ15N values equivalent to or in a lower range than those of horse specimens (Equus sp.). We expanded our preliminary dataset to a larger sampling size of mammoth, other large herbivores, and carnivores from contemporaneous and nearby sites of Buzhanka 2, Eliseevichi, and Yudinovo. The unusual low mammoth δ15N values were confirmed at Buzhanka 2 and for some specimens from Eliseevichi, while most individuals from Yudinovo displayed the expected high δ15N values, meaning similar to those of the large canids. The possibility of a contrast in migration pattern is not supported since the δ34S values, a marker of mobility, do not correlate with the δ15N values of mammoth bone collagen. No clear chronological tendency could be revealed, at least not at the scale of radiocarbon dating. The low range in δ15N values is likely to reflect a change in the specific niche of the mammoth in the southern part of its distribution.
Although radiocarbon accelerator mass spectrometry (14C AMS) surpasses conventional radiometric methods in many aspects, they still represent an interesting alternative, especially for studies unconstrained by sample size. Here we showed that the gas proportional counting technique can be used for bone samples, processed only by a simple ABA method, and ethanol, distilled from wine samples. The feasibility of the described methods was verified by successful dating of 11 well-preserved vertebrate bones of modern to 21 kyr BP age excavated from different caves in Slovakia from which collagen was also extracted, as well as by determination of 14C concentration in two modern western Slovakia vintages, which matches well the atmospheric Δ14C level for the respective region and grape vegetation period. Various empiric factors affecting the yield of the thoroughly tested procedures used for processing of samples and their optimization parameters are discussed as well.
Radiation therapy, widely used in the treatment of a variety of malignancies in the pelvic area, is associated with inevitable damage to the surrounding healthy tissues. We have applied atomic force microscopy (AFM) to track the early damaging effects of ionizing radiation on the collagen structures in the experimental animals’ bladder and rectum. The first signs of the low-dose radiation (2 Gy) effect were detected by AFM as early as 1 week postirradiation. The observed changes were consistent with initial radiation destruction of the protein matrix. The alterations in the collagen fibers’ packing 1 month postirradiation were indicative of the onset of fibrotic processes. The destructive effect of higher radiation doses was probed 1 day posttreatment. The severity of the radiation damage was proportional to the dose, from relatively minor changes in the collagen packing at 8 Gy to the growing collagen matrix destruction at higher doses and complete three-dimensional collagen network restructuring towards fibrotic-type architecture at the dose of 22 Gy. The AFM study appeared superior to the optical microscopy-based studies in its sensitivity to early radiation damage of tissues, providing valuable additional information on the onset and development of the collagen matrix destruction and remodeling.
In this paper, first results comparing modified Longin and ninhydrin collagen extraction methodologies are presented. The goal of this study is to investigate the bones of several species with different ages, preservation conditions, and collagen contents to determine the most suitable preparation method. Different types of samples are used such as VIRI samples, previously dated bones, and background samples. Each bone has undergone elemental analysis, infrared analysis, and 14C measurement. The results are presented and the advantages and disadvantages of each preparation method are discussed. In general, results obtained by the two methods are in accordance with the consensus value for 2σ uncertainty. For VIRI I and a mammoth bone, the ninhydrin preparation gives, respectively, 8450±70 BP and 14,870±60 BP whereas the modified Longin process gives 8365±45 BP and 14,750±100 BP in agreement with the expected values. From the experimental point of view, the modified Longin process is easier to implement than the ninhydrin protocol. From this approach, we can conclude that the modified Longin process could be preferred in most cases and particularly when the amount of bone is small and the sample is not too contaminated.
Wild boar and roe deer samples from an excavation in Dispilio, Greece, were subjected to collagen extraction protocols to reconstruct the paleoecological regime. Radiocarbon (14C) analysis suggested the Middle/Late Neolithic period and the database was updated with collagen samples. The 14C model concluded to a possible local deforestation effect in the settlement subbasin confirmed by sediment δ13C and δ15N values. Carbon isotope values in collagen samples concluded in C3 plant type. Both carbon and nitrogen isotopes indicated the differences in dietary habits and/or metabolic system between the two Late Neolithic I species. Roe deer samples were classified as purely herbivorous. δ15N values of wild boar collagen samples from Dispilio reflected a diet mainly characterized by terrestrial protein. Compared to literature data, wild boar samples from the Dispilio excavation concluded that the animals might have lived close to the settlement where their diet could be supplemented by a consistent animal protein fraction. Finally, it is concluded that rainfall is an important factor that affects plant, and consequently animal, δ15N values. Therefore, the rainfall regime should always be considered in paleodietary studies.
Our previous study demonstrated that supplemental psyllium fibre increased cytoprotective heat-shock protein (Hsp) 25 levels in the intestinal cells of mice. Here, we examined the effect of psyllium fibre on colonic gene and protein expression and faecal microbiota in normal and colitic mice to improve the understanding of the preventive role of the supplement. DNA microarray analysis revealed that a 10 % psyllium fibre diet administered for 5 d up-regulated eleven extracellular matrix (ECM)-associated genes, including collagens and fibronectins, in normal mice. Acute colitis was induced using dextran sodium sulphate (DSS) in mice that were administered a pre-feeding 5 to 10 % psyllium fibre diet for 5 d. Psyllium fibre partially ameliorated or resolved the DSS-induced colon damage and inflammation characterised by body weight loss, colon shortening, increased levels of pro-inflammatory cytokines and decreased tight junction protein expression in the colon. Analysis of faecal microbiota using denaturing gradient gel electrophoresis of the PCR-amplified 16S rRNA gene demonstrated that psyllium fibre affected the colonic microbiota. Intestinal permeability was evaluated by growing intestinal Caco-2 cell monolayers on membrane filter supports coated with or without fibronectin and collagen. Cells grown on collagen and fibronectin coating showed higher transepithelial electrical resistance, indicating a strengthening of barrier integrity. Therefore, increased Hsp25 levels and modification of colonic ECM contribute to the observed psyllium-mediated protection against DSS-induced colitis. Furthermore, ECM modification appears to play a role in the strengthening of the colon barrier. In conclusion, psyllium fibre may be useful in the prevention of intestinal inflammatory diseases.
Fibrillar collagen in tendons and its natural development in rabbits are discussed in this paper. Achilles tendons from newborn (~7 days) to elderly (~38 months) rabbits were monitored in intact (ntendons=24) and microtome sectioned (ntendons=11) states with label-free second harmonic generation microscopy. After sectioning, the collagen fiber pattern was irregular for the younger animals and remained oriented parallel to the load axis of the tendon for the older animals. In contrast, the collagen fiber pattern in the intact samples followed the load axis for all the age groups. However, there was a significant difference in the tendon crimp pattern appearance between the age groups. The crimp amplitude (A) and wavelength (Λ) started at very low values (A=2.0±0.6 µm, Λ=19±4 µm) for the newborn animals. Both parameters increased for the sexually mature animals (>5 months old). When the animals were fully mature the amplitude decreased but the wavelength kept increasing. The results revealed that the microtome sectioning artifacts depend on the age of animals and that the collagen crimp pattern reflects the physical growth and development.
Due to increased longevity, women can expect to live more than one-third of their lives in a post-menopausal state, which is characterised by low circulating levels of oestrogen and progesterone. The aim of this review is to provide insights into current knowledge of the effect of female hormones (or lack of female hormones) on skeletal muscle protein turnover at rest and in response to exercise. This review is primarily based on data from human trials. Many elderly post-menopausal women experience physical disabilities and loss of independence related to sarcopenia, which reduces life quality and is associated with substantial financial costs. Resistance training and dietary optimisation can counteract or at least decelerate the degenerative ageing process, but lack of oestrogen in post-menopausal women may reduce their sensitivity to these anabolic stimuli and accelerate muscle loss. Tendons and ligaments are also affected by sex hormones, but the effect seems to differ between endogenous and exogenous female hormones. Furthermore, the effect seems to depend on the age, and as a result influence the biomechanical properties of the ligaments and tendons differentially. Based on the present knowledge oestrogen seems to play a significant role with regard to skeletal muscle protein turnover. Therefore, oestrogen/hormonal replacement therapy may counteract the degenerative changes in skeletal muscle. Nevertheless, there is a need for greater insight into the direct and indirect mechanistic effects of female hormones before any evidence-based recommendations regarding type, dose, duration and timing of hormone replacement therapy can be provided.
Insoluble bone collagen is one of the most common materials used for high-resolution radiocarbon (14C) dating. Unfortunately, in some bones, poor preservation of the insoluble collagen excludes the possibility of dating. During the burial of the bone the collagen sometimes degrades into peptides. These peptides are soluble in the acid used to dissolve the bone mineral. It is known that under appropriate conditions, collagen has the ability to self-assemble. Here we exploit this capability and present a method for reassembling the soluble collagen peptides in archaeological bones and dating them. We treated the acid fraction generated during the demineralization of the bone by desalting and neutralizing the solution by dialysis. During the dialysis, the soluble collagen peptides reassemble and precipitate in the dialysis bag. We used FTIR spectroscopy to determine that the precipitated material is indeed collagen. The 14C dates obtained from the reassembled collagen were compared to the dates of “standard” insoluble collagen, extracted in parallel from the same bone. Although there are some divergences of the dates, 3 out of 10 samples could have been dated only by the reassembled collagen. This shows that collagen peptides reassembly can be a valuable tool for dating bones with little or no insoluble collagen.
Multi-layer reconstruction has become standard in endoscopic skull base surgery. The inlay component used can vary among autografts, allografts, xenografts and synthetics, primarily based on surgeon preference. The short- and long-term outcomes of collagen matrix in skull base reconstruction are described.
A case series of patients who underwent endoscopic skull base reconstruction with collagen matrix inlay were assessed. Immediate peri-operative outcomes (cerebrospinal fluid leak, meningitis, ventriculitis, intracranial bleeding, epistaxis, seizures) and delayed complications (delayed healing, meningoencephalocele, prolapse of reconstruction, delayed cerebrospinal fluid leak, ascending meningitis) were examined.
Of 120 patients (51.0 ± 17.5 years, 41.7 per cent female), peri-operative complications totalled 12.7 per cent (cerebrospinal fluid leak, 3.3 per cent; meningitis, 3.3 per cent; other intracranial infections, 2.5 per cent; intracranial bleeding, 1.7 per cent; epistaxis, 1.7 per cent; and seizures, 0 per cent). Delayed complications did not occur in any patients.
Collagen matrix is an effective inlay material. It provides robust long-term separation between sinus and cranial cavities, and avoids donor site morbidity, but carries additional cost.