Collagen Composition Research Articles

Evaluating the efficacy of collagen isolation using stable isotope analysis and infrared spectroscopy

Stable isotope analysis of bone and dental collagen is one of the most common methods to investigate the ecology of modern and extinct human and animal populations. However, since bone and dentine are composite materials with both organic and mineral components, the mineral component must be removed prior to analysis. In this study we investigated the timing and efficacy of mineral removal from bone and dentine. We performed a series of time-step experiments that show that mineral removal can be quantified over short periods of time using Fourier Transform Infrared Spectroscopy (FTIR), and collagen alteration can be tracked using a combination of stable isotope analysis and elemental analysis. We tested our methods on three modern materials: mammalian bone, mammalian dentine, and shark dentine. Our results show: 1) mineral removal is a necessary step, as structural carbonate has a strong influence on stable isotope compositions; 2) demineralization using weak acid (0.1 M HCL) does not appear to alter the elemental and isotopic compositions of collagen. Our methods can be used as a framework to evaluate the need-for and efficacy of other demineralization methods in use today including EDTA-demineralization and lipid removal.

Assessment of Collagen in Translational Models of Lung Research.

The extracellular matrix (ECM) plays an important role in lung health and disease. Collagen is the main component of the lung ECM, widely used for the establishment of in vitro and organotypic models of lung disease, and as scaffold material of general interest for the field of lung bioengineering. Collagen also is the main readout for fibrotic lung disease, where collagen composition and molecular properties are drastically changed and ultimately result in dysfunctional "scarred" tissue. Because of the central role of collagen in lung disease, quantification, determination of molecular properties, and three-dimensional visualization of collagen is important for both development and characterization of translational models of lung research. In this chapter, we provide a comprehensive overview on the various methodologies currently available for quantification and characterization of collagen including their detection principles, advantages, and disadvantages.

Prospects for the Use of the Swim Bladder of the Silver Carp and the Possibility of its Application in Medicine

Fish waste can serve as a raw material for medical devices. Matrices for tissue regeneration are currently in great demand in medicine, which can be obtained from the walls of the swim bladder of fish, which has a collagen composition that has properties that are inert to recipients. The most suitable in terms of overall, morphological, biological properties for the manufacture of scaffolds is the wall of the anterior chamber of the swim bladder of the silver carp, which is widespread and is an object of aquaculture in our country. In the course of this work, for the first time, the size of the anterior chamber of the swim bladder was measured in silver carp of different weight categories. According to the results of the study, the dependence of the thickness and diameter of the surface of the anterior chamber of the swim bladder on the weight of the silver carp was established, which can be very useful in selecting the material according to the required criteria for the manufacture of implantable medical plates.

Deciphering the Role of TGF-β1 in Altering Collagen I and Collagen III in the New Zealand Rabbit's (Oryctolagus cuniculus) Urethral Wall in Urethral Stricture Development.

Presently, there's a lack of standardization in animal models used for studying urethral stricture. Transforming Growth Factor Beta 1 (TGF-β1) is known to regulate the deposition of extracellular matrix in both normal and pathological conditions. This factor holds promise as a potential model for simulating urethral stricture. This study aims to investigate the impact of Transforming Growth Factor Beta 1 (TGF-β1) on Collagen I and Collagen III within the urethral wall of New Zealand Rabbits (Oryctolagus cuniculus) in the context of developing urethral stricture in animal models. We conducted genuine laboratory experiments using Male New Zealand rabbits (Oryctolagus cuniculus), which were categorized into five groups: control, placebo, and three treatment groups (TGF-β1 injections of 1 µg, 2 µg, 4 µg). After a duration of 6 weeks, we conducted urethrography, histopathological analysis, and assessed the formation of collagen I and collagen III within the urethral wall. Elevating the dosage of TGF-β1 led to a reduction in the average urethral lumen diameter of rabbits (29.3% in the 2µg group and 34% in the 4µg group) compared to the control group. In fact, three rabbits experienced a decrease of ≤ 50% in their urethral lumen diameter. As the doses of TGF-β1 increased, we observed significant increases in the density of collagen I, and collagen III in both the periluminal and peripheral regions of the urethral spongiosum. Additionally, there was a tendency for the collagen I/collagen III ratio to decrease in the periluminal region, with collagen III density surpassing that of collagen I. In the peripheral spongiosa area, notable mean differences were observed between the control group, 1T, and 2T groups, with collagen I density tending to be higher than that of collagen III. Furthermore, the percentage of urethral lumen diameter exhibited a robust negative correlation with periluminal collagen I density (r = -0.672, p = 0.001), peripheral spongiosa collagen I density (r = -0.603, p = 0.005), periluminal collagen III density (r = -0.717, p = 0.001), and an exceptionally strong negative correlation with collagen III density of peripheral spongiosa (r = -0.804, p = 0.000). TGF-β1 exerts an influence on altering the composition of collagen I and collagen III within the urethral wall of rabbits, leading to a reduction in the diameter of the urethral lumen. Further research is warranted to determine the optimal dose of TGF-β1 required to induce urethral stricture effectively.

TGF-β1 Effects on Total Collagen of the New Zealand Rabbit's Urethral Wall (Oryctolagus cuniculus) in Animal Models of Urethral Stricture.

Currently, animal models of urethral stricture are not standardized. Transforming Growth Factor Beta 1 (TGF-β1) regulates extracellular matrix deposition in homeostatic and pathological responses. The aim of this study was to present the potential model to be developed as a urethral stricture. True experimental laboratory research was conducted by using Male New Zealand rabbits (Oryctolagus cuniculus), which were divided into 5 groups; control, placebo, and 3 treatment groups (TGF-β1 injection of 1 µg, 2 µg, 4 µg). Urethrography, histopathological analysis, and evaluation of total collagen formation of the urethral wall were performed after 6 weeks. An increase in the dose of TGF-β1 decreased the mean rabbit's urethral lumen diameter (29.3% in the 2µg group and 34% in the 4µg group) compared to controls. Three rabbits decreased as much as ≤ 50% in urethral lumen diameter. Significant increases in total collagen density in the periluminal and peripheral urethral spongiosum were noted by increasing doses of TGF-β1. The percentage of urethral lumen diameter has a strong negative correlation with periluminal total collagen density (r = -0,798; p = 0,000) and very strong negative correlation with peripheral spongiosa total collagen density (r = -0,748, p = 0,000). TGF-β1 plays a role in changing total collagen compositions of the rabbit's urethral wall, decreasing the urethral lumen diameter. Further research with increasing doses of TGF-β1 is needed to determine the effective dose of TGF-β1 in inducing urethral stricture.

Regional and disease specific human lung extracellular matrix composition

Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), are characterized by regional extracellular matrix (ECM) remodeling which contributes to disease progression. Previous proteomic studies on whole decellularized lungs have provided detailed characterization on the impact of COPD and IPF on total lung ECM composition. However, such studies are unable to determine the differences in ECM composition between individual anatomical regions of the lung. Here, we employ a post-decellularization dissection method to compare the ECM composition of whole decellularized lungs (wECM) and specific anatomical lung regions, including alveolar-enriched ECM (aECM), airway ECM (airECM), and vasculature ECM (vECM), between non-diseased (ND), COPD, and IPF human lungs. We demonstrate, using mass spectrometry, that individual regions possess a unique ECM signature characterized primarily by differences in collagen composition and basement-membrane associated proteins, including ECM glycoproteins. We further demonstrate that both COPD and IPF lead to alterations in lung ECM composition in a region-specific manner, including enrichment of type-III collagen and fibulin in IPF aECM. Taken together, this study provides methodology for future studies, including isolation of region-specific lung biomaterials, as well as a dataset that may be applied for the identification of novel ECM targets for therapeutics.

In utero exposure to selective serotonin re-uptake inhibitor affects murine mandibular development.

Antidepressants, specifically Selective Serotonin Re-uptake Inhibitors (SSRIs), that alter serotonin metabolism are currently the most commonly prescribed drugs for the treatment of depression. There is some evidence to suggest these drugs contribute to birth defects. As jaw development is often altered in craniofacial birth defects, the purpose of this study was to interrogate the effects of in utero SSRI exposure in a preclinical model of mandible development. Wild-type C57BL6 mice were used to produce litters that were exposed in utero to an SSRI, Citalopram (500 μg/day). Murine mandibles from P15 pups were analysed for a change in shape and composition. Analysis indicated an overall shape change with total mandibular length and ramus height being shorter in exposed pups as compared to controls. Histomorphometric analysis revealed that first molar length was longer in exposed pups while third molar length was shorter in exposed as compared to control. Histological investigation of molars and surrounding periodontium revealed no change in collagen content of the molar in exposed pups, some alteration in collagen composition in the periodontium, increased alkaline phosphatase in molars and periodontium and decreased mesenchymal cell marker presence in exposed mandibles. The results of this study reveal SSRI exposure may interrupt mandible growth as well as overall dental maturation in a model of development giving insight into the expectation that children exposed to SSRIs may require orthodontic intervention.

Effects of consolidants and their removal by polar solvents on the stable isotope compositions of bone

Although conservation practices facilitate the preservation of museum collections, procedures used to stabilize ancient materials can alter their chemical and isotopic compositions, impacting the viability of such specimens for life history investigations. In this study, we applied two common consolidants – polyvinyl acetate (PVAc) and acrylic resin (Paraloid B-72™) – followed by two consolidant removal procedures to assess the impacts of these processes on the stable isotope compositions and structural characteristics of a series of subsamples taken from a representative archaeological faunal bone. We examined the collagen (δ13Ccol and δ15Ncol), structural carbonate (δ13Csc and δ18Osc), and phosphate (δ18Op) isotopic compositions of the bone sample before and after consolidation to evaluate consolidant removal procedures. We also measured δ13Csc, δ18Osc, and structural characteristics before and after the pre-treatment commonly used to remove organic matter and secondary carbonate prior to isotopic analyses.Our results produced five main outcomes. First, an acetone treatment shorter than 48 h was sufficient to remove PVAc and acrylic resin from archaeological bone without altering the original δ13Ccol and δ15Ncol. Second, prolonged exposure (>48 h) to polar solvents during the consolidant removal procedure and collagen extraction modified collagen isotopic compositions, lowering its original δ13Ccol by 0.3–0.4 ‰ and increasing its original δ15Ncol by up to 0.9 ‰. Third, the consolidant application and removal procedures altered the original δ13Csc and δ18Osc. Fourth, the change in δ18Osc was likely caused by the bleach (2% NaClO at 20 °C for 72 h)/acetic acid (0.1 M for 4 h at 20 °C) pre-treatment applied prior to isotopic analyses. Fifth, our results confirm and expand on previous studies reporting that δ18Op remains unaltered after consolidant and solvent procedures. These outcomes provide a framework for obtaining reliable stable isotope compositions from archaeological bones that have been stabilized using consolidants.

Synchronous change in the intensified millet cultivation and ecological environment from the early to middle Holocene on the Inner Mongolia Plateau, northern China

The mechanisms of the origin and dispersal of millet agriculture in northern China are poorly understood. We used plant macroremains, stable isotope compositions of human bone collagen, and pollen records from the Sitai site to reconstruct changes in subsistence strategies and their relationship with the ecological environment from the early to middle Holocene on the Inner Mongolian Plateau in northern China. Charred weed-like seeds, the bones of small mammals, eggshell fragments, together with microliths, indicate the practice of hunter-gatherer subsistence strategies during 10,500–10,200 cal yr BP. Deciduous broadleaved forest-steppe vegetation was present around the Sitai site during the early middle Holocene (8000–7000 cal yr BP). Additionally, isotopic compositions of human bones and plant remains reveal that millet agriculture and hunting-gathering appeared in the early middle Holocene. The spread of millet agriculture on the Inner Mongolian Plateau was likely favored by an increase in precipitation between 8000 and 7000 cal yr BP. The development of millet agriculture on the Inner Mongolia Plateau and the Loess Plateau was the prelude to its subsequent spread to the Tibet Plateau.

Phosphorylation of CaMK and CREB-Mediated Cardiac Aldosterone Synthesis Induced by Arginine Vasopressin in Rats with Myocardial Infarction

Both aldosterone and arginine vasopressin (AVP) are produced in the heart and may participate in cardiac fibrosis. However, their relationship remains unknown. This study aims to demonstrate the regulation and role of AVP in aldosterone synthesis in the heart. Rats were subjected to a sham operation or myocardial infarction (MI) by ligating the coronary artery. Cardiac function and fibrosis were assessed using echocardiography and immunohistochemical staining, respectively. In addition, the effects of AVP stimulation on cardiac microvascular endothelial cells (CMECs) were studied using ELISA, real-time PCR, and Western blotting. Compared with the rats having undergone a sham operation, the MI rats had an increased LVMI, type I collagen composition, and concentrations of aldosterone and AVP in the heart but decreased cardiac function. As the MI rats aged, the LVMI, type I collagen, aldosterone, and AVP increased, while the LVMI decreased. Furthermore, AVP time-dependently induced aldosterone secretion and CYP11B2 mRNA expression in CMECs. The p-CREB levels were significantly increased by AVP. Nevertheless, these effects were completely blocked by SR49059 or partially inhibited by KN93. This study demonstrated that AVP could induce the secretion of local cardiac aldosterone, which may involve CaMK and CREB phosphorylation and CYP11B2 upregulation through V1 receptor activation.

Combining δ 13C and δ 15N from bone and dentine in marine mammal palaeoecological research: insights from toothed whales

ABSTRACT Stable carbon (δ 13C) and nitrogen (δ 15N) isotopic compositions of bone and dentine collagen extracted from museum specimens have been widely used to study the paleoecology of past populations. Due to possible systematic differences in stable isotope values between bone and dentine, dentine values need to be transformed into bone-collagen equivalent using a correction factor to allow comparisons between the two collagen sources. Here, we provide correction factors to transform dentine δ 13C and δ 15N values into bone-collagen equivalent for two toothed whales: narwhal and beluga. We sampled bone and dentine from the skulls of 11 narwhals and 26 belugas. In narwhals, dentine was sampled from tusk and embedded tooth; in belugas, dentine was sampled from tooth. δ 13C and δ 15N were measured, and intra-individual bone and dentine isotopic compositions were used to calculate correction factors for each species. We detected differences in δ 13C and δ 15N. In both narwhals and belugas, we found lower average δ 13C and δ 15N in bone compared with dentine. The correction factors provided by the study enable the combined analysis of stable isotope data from bone and dentine in these species.

Quantative MRI predicts tendon mechanical behavior, collagen composition, and organization.

Quantitative magnetic resonance imaging (qMRI) measures have provided insights into the composition, quality, and structure-function of musculoskeletal tissues. Low signal-to-noise ratio has limited application to tendon. Advances in scanning sequences and sample positioning have improved signal from tendon allowing for evaluation of structure and function. The purpose of this study was to elucidate relationships between tendon qMRI metrics (T1, T2, T1ρand diffusion tensor imaging [DTI] metrics) with tendon tissue mechanics, collagen concentration and organization. Sixteen human Achilles tendon specimens were collected, imaged with qMRI, and subjected to mechanical testing with quantitative polarized light imaging. T2 values were related to tendon mechanics [peak stress (rsp = 0.51, p = 0.044), equilibrium stress (rsp = 0.54, p = 0.033), percent relaxation (rsp = -0.55, p = 0.027), hysteresis (rsp = -0.64, p = 0.007), linear modulus (rsp = 0.67, p = 0.009)]. T1ρhad a statistically significant relationship with percent relaxation (r = 0.50, p = 0.048). Collagen content was significantly related to DTI measures (range of r = 0.56-0.62). T2 values from a single slice of the midportion of human Achilles tendons were strongest predictors of tendon tensile mechanical metrics. DTI diffusivity indices (mean diffusivity, axial diffusivity, radial diffusivity) were strongly correlated with collagen content. These findings build on a growing body of literature supporting the feasibility of qMRI to characterize tendon tissue and noninvasively measure tendon structure and function. Statement of Clinical Significance: Quantitative MRI can be applied to characterize tendon tissue and is a noninvasive measure that relates to tendon composition and mechanical behavior.

Lipid nanoparticle-assisted miR29a delivery based on core-shell nanofibers improves tendon healing by cross-regulation of the immune response and matrix remodeling

Inferior healing and peritendinous adhesions are the major clinical problems following Achilles tendon injury, leading to impaired motor function and an increased risk of re-rupture. These complications are presumed to be inextricably linked to inflammation and fibroscar formation. Here, microRNA29a is identified as a promising therapeutic target for tendon injury through the cross-regulation of the immune response and matrix remodeling. MiR29a-LNPs were successfully prepared by microfluidic technology. They are then loaded into the core-shell nanofibers to achieve local delivery in the injured tendon, where the shell layer is composed of PELA for anti-adhesion. Our studies reveal that miR29a regulates collagen synthesis and NF-κB activation in tenocytes, and promotes macrophage polarization by inhibiting the inflammasome pathway. In vivo studies of the Achilles tendon-rupture model indicate the best repair in the miR29a group, as evidenced by superior collagen composition and alignment, higher mechanical strength, and better functional recovery. In conclusion, a functionalized anti-adhesive membrane that promotes nascent tendon matrix remodeling and improves the regenerative immune microenvironment is developed for the treatment of tendon injury.

Periostin modulates extracellular matrix behavior in tendons

Periostin, originally named osteoblast-specific factor 2 (OSF-2) has been identified primarily in collagen rich, biomechanically active tissues where its role has been implicated in mechanisms to maintain the extracellular matrix (ECM), including collagen fibrillogenesis and crosslinking. It is well documented that periostin plays a role in wound healing and scar formation after injury, in part, by promoting cell proliferation, myofibroblast differentiation, and/or collagen fibrillogenesis. Given the significance of periostin in other scar forming models, we hypothesized that periostin will influence Achilles tendon healing by modulating ECM production. Therefore, the objective of this study was to elucidate the effects of periostin during Achilles tendon healing using periostin homozygous (Postn−/−) and heterozygous (Postn+/-) mouse models. A second experiment was included to further examine the influence of periostin on collagen composition and function using intact dorsal tail tendons. Overall, Postn−/− and Postn+/- Achilles tendons exhibited impaired healing as demonstrated by delayed wound closure, increased type III collagen production, decreased cell proliferation, and reduced tensile strength. Periostin ablation also reduced tensile strength and stiffness, and altered collagen fibril distribution in the intact dorsal tail tendons. Achilles tendon outcomes support our hypothesis that periostin influences healing, while tail tendon results indicate that periostin also affects ECM morphology and behavior in mouse tendons.

Only Acute but Not Chronic Thrombocytopenia Protects Mice against Left Ventricular Dysfunction after Acute Myocardial Infarction.

Platelets are major players of thrombosis and inflammation after acute myocardial infarction (AMI). The impact of thrombocytopenia on platelet-induced cellular processes post AMI is not well defined. The left anterior descending artery was ligated in C57/Bl6 mice and in two thrombocytopenic mouse models to induce AMI. Platelets from STEMI patients and from C57/Bl6 mice displayed enhanced platelet activation after AMI. This allows platelets to migrate into the infarct but not into the remote zone of the left ventricle. Acute thrombocytopenia by antibody-induced platelet depletion resulted in reduced infarct size and improved cardiac function 24 h and 21 days post AMI. This was due to reduced platelet-mediated inflammation after 24 h and reduced scar formation after 21 days post AMI. The collagen composition and interstitial collagen content in the left ventricle were altered due to platelet interaction with cardiac fibroblasts. Acute inflammation was also significantly reduced in Mpl-/- mice with chronic thrombocytopenia, but cardiac remodeling was unaltered. Consequently, left ventricular function, infarct size and scar formation in Mpl-/- mice were comparable to controls. This study discovers a novel role for platelets in cardiac remodeling and reveals that acute but not chronic thrombocytopenia protects left ventricular function post AMI.

Geographic origin and social status of the Gallic warriors from Ribemont‐sur‐Ancre (France) studied through isotope systematics of bone remains

AbstractRibemont‐sur‐Ancre, France, is the biggest excavated human skeletal assemblage of Northern Gaul. It is interpreted as a post‐battle deposit constructed to celebrate war. However, the chronology of the site, its organization and the origin of the buried warriors are still poorly understood. These problems are studied by a multi‐proxy approach, based on radiocarbon dating, stable and radiogenic isotope analysis of bone remains from three structures within the site. A total of 47 human and 12 animal bone samples were analyzed for their oxygen isotope composition of bone bioapatite (δ18Op), carbon and nitrogen isotope compositions of bone collagen (δ13Ccoll and δ15Ncoll). Subsamples of 17 bone collagen and 20 bone samples were selected for radiocarbon dating and strontium isotope analysis (87Sr/86Sr), respectively. All samples show preservation from diagenesis, although 87Sr/86Sr contribution from the burial environment cannot be excluded. Therefore, the strontium isotope data are included in the study but are not used in the interpretation of the results. Homogeneous radiocarbon dates of human remains are observed, whereas later dates are obtained for some animal remains, highlighting the continuous use of the monument decades after the conflict. Human individuals have homogeneous δ18Op values, which are consistent with independent numismatic analyses and lead us to propose the Maine region as a potential geographic origin. Heterogeneities of δ13Ccoll and δ15Ncoll values are highlighted between individuals from different structures and are attributed to preferential access to animal protein and different social statuses among the buried warriors.

Penuaan Kulit dan Terapi yang Aman Bagi Geriatri: Artikel Review

Geriatric have different skin characteristic compared with they who are in young age. There is a change in both intrinsic and extrinsic aging factors causing skin aging which is marked by skin wrinkles, loss of elasticity, and textural changes. The composition of collagen in geriatrics has decreased and there is an increase in matrix metalloproteinases resulted in skin aging. These conditions together with exposure to UVR, infection, and other risk factors cause various skin problems such as xerosis cutis, decubitus ulcers, dermatophytosis, and herpes zoster. Skin problems can be prevented by changing lifestyles, using some cosmetics, medications, and having treatments. The prevention methods continues to grow and is still being researched to get optimal results and best suit skin conditions. Treatment of skin diseases in geriatrics should be carried out with caution considering the multimorbidity and immunosuppression that often occur in geriatrics. This article discusses skin differences in geriatrics, mechanisms of skin aging, common skin diseases, prevention, and safe treatment in geriatrics.

Collagen cross-link profiles and mineral are different between the mandible and femur with site specific response to perturbed collagen

Compromises to collagen and mineral lead to a decrease in whole bone quantity and quality in a variety of systemic diseases, yet, clinically, disease manifestations differ between craniofacial and long bones. Collagen alterations can occur through post-translational modification via lysyl oxidase (LOX), which catalyzes enzymatic collagen cross-link formation, as well as through non-enzymatic advanced glycation end products (AGEs) such as pentosidine and carboxymethyl-lysine (CML). Characterization of the cross-links and AGEs, and comparison of the mineral and collagen modifications in craniofacial and long bones represent a critical gap in knowledge. However, alterations to either the mineral or collagen in bone may contribute to disease progression and, subsequently, the anatomical site dependence of a variety of diseases. Therefore, we hypothesized that collagen cross-links and AGEs differ between craniofacial and long bones and that altered collagen cross-linking reduces mineral quality in an anatomic location dependent. To study the effects of cross-link inhibition on mineralization between anatomical sites, beta-aminoproprionitrile (BAPN) was administered to rapidly growing, 5–8 week-old male mice. BAPN is a dose-dependent inhibitor of LOX that pharmacologically alters enzymatic cross-link formation. Long bones (femora) and craniofacial bones (mandibles) were compared for mineral quantity and quality, collagen cross-link and AGE profiles, and tissue level mechanics, as well as the response to altered cross-links via BAPN. A highly sensitive liquid chromatography/mass spectrometry (LC-MS) method was developed which allowed for quantification of site-dependent accumulation of the advanced glycation end-product, carboxymethyl-lysine (CML). CML was ∼8.3× higher in the mandible than the femur. The mandible had significantly higher collagen maturation, mineral crystallinity, and Young's modulus, but lower carbonation, than the femur. BAPN also had anatomic specific effects, leading to significant decreases in mature cross-links in the mandible, and an increase in mineral carbonation in the femur. This differential response of both the mineral and collagen composition to BAPN between the mandible and femur highlights the need to further understand how inherent compositional differences in collagen and mineral contribute to anatomic-site specific manifestations of disease in both craniofacial and long bones.

Hyperglycaemia-induced degeneration of bioprosthetic valve tissue and the pivotal role of the extracellular matrix molecule biglycan

Abstract Background/Introduction Bioprosthetic valve degeneration is a growing clinical challenge in our aging society. Especially type 2 diabetes mellitus represents a risk factor for bioprosthetic valve deterioration with likewise worldwide rising prevalence. The proteoglycan biglycan has been shown to be associated with degenerative changes of the valve in diabetic patients even though underlying mechanisms are yet unknown. Purpose The impact of hyperglycaemia on bioprosthetic valve tissue was analysed in an established mouse model of ectopic calcification. Thereby, the role of biglycan in bioprosthetic valve degeneration was assessed in biglycan-deficient mice (Bgn-/0) in comparison to wild type littermates (WT). Methods Hyperglycaemia was induced in six weeks old mice by intraperitoneal injection of streptozotocin (STZ; 55 mg/kg body weight) on five consecutive days. Blood glucose concentration was verified two weeks after treatment and bioprosthetic valve material was implanted subcutaneously. After eight weeks, phenotype of invading cells and extracellular matrix remodelling of implants were analysed with histochemical and immunohistological staining. Furthermore, gene expression of invading cells, circulating cytokines in blood plasma and calcium accumulation in the tissue were quantified. Results STZ treatment significantly increased blood glucose levels in both genotype groups (WT-STZ: 156 mg/dl; WT+STZ: 353 mg/dl; Bgn-/0-STZ: 142 mg/dl; Bgn-/0+STZ: 396 mg/dl). Immunohistological staining identified most of cells invading the bioprosthetic tissue and the surrounded capsule as Mac2-positive and partly positive for vimentin, whereas alpha smooth muscle actin and von Willebrand factor were only detectable sporadically. Movat pentachrome staining exhibited an altered extracellular matrix composition of collagen and proteoglycans due to hyperglycaemia, while the elastin amount remained stable. Gene expression analysis showed an increased expression of the proteoglycan decorin in hyperglycaemic mice (WT-STZ: 0.55; WT+STZ: 1.13; Bgn-/0-STZ: 0.51; Bgn-/0+STZ: 1.70). Circulating cytokines (IL-1β, IL-2, IL-5, IL-10, INF-γ, TNF-α, GM-CSF) were influenced neither by hyperglycaemia nor by the genotype of the mice. Alizarin and von Kossa staining exhibited small to moderate calcium deposits in the bioprosthetic valve tissue. A colorimetric assay showed a significantly increased calcium accumulation in WT with hyperglycaemia versus normoglycemic WT (WT-STZ: 8.96 μg/mg; WT+STZ: 18.54 μg/mg), an observation that was lost in Bgn-/0 (Bgn-/0-STZ: 9.84 μg/mg; Bgn-/0+STZ: 12.97 μg/mg). Conclusion Hyperglycaemia significantly promotes destructive remodelling in bioprosthetic valve material. Biglycan-deficiency limits degenerative processes associated with diabetes, suggesting that biglycan as a component of the extracellular matrix has an adverse effect in diabetes-associated bioprosthetic valve deterioration. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Dr. Rusche grant of the German Heart Foundation and the German Society for Thoracic and Cardiovascular Surgery

Inflammatory Changes and Composition of Collagen during Cervical Ripening in Cows.

Simple SummaryOne cause of parturition abnormalities in cows may be the insufficient opening of the birth canal; however, the process by which the canal becomes flexible in cows is unknown. Herein, we observed changes in the cervix from late pregnancy to parturition to clarify the physiological changes related to cervical ripening during normal calving. Mucus and tissue samples from the cervix were collected from 41 cows at 30-day intervals from 200 to 260 days of gestation and at 7-day intervals from 260 days of gestation to parturition. The percentage of neutrophils in the mucus (PMN%) was calculated, and the concentration of interleukin (IL)-8, an inflammatory cytokine, was measured. The cervical tissues were observed under a microscope, and the percentage of type I collagen was calculated. The PMN% in the cervical mucus increased 5 weeks before delivery, peaking 1 week before calving; IL-8 was significantly increased at 295 days compared to that at 200 days of gestation. The percentage of type I collagen in the cervical tissue decreased significantly from 200 days to 274 days of pregnancy and continued to decrease thereafter until the week of calving. In conclusion, the cervix of cows begins to ripen at approximately 260 days of pregnancy.Dystocia and stillbirths in cows pose a high risk of loss of both dams and fetuses, thereby resulting in high economic losses. One of the causes of these problems is birth canal abnormalities. Thus, to prevent these occurrences, it is necessary to understand the mechanisms underlying cervical ripening. Although physiological inflammatory responses and changes in collagen composition have been reported in humans and mice, related information is scarce for cows. We observed inflammatory changes and changes in the collagen composition in the cervix from late pregnancy to parturition to clarify some of the physiological changes associated with cervical ripening during normal calving in cows. Cervical mucus and tissue samples were collected from 41 Japanese Black cows at 200, 230, and 260 days of gestation and at 7-day intervals thereafter until parturition. The percentage of polymorphonuclear neutrophils (PMN%) in the mucus was calculated, and interleukin (IL)-8 concentration was determined by enzyme-linked immunosorbent assay. Blood samples were collected from the jugular vein, and leukocyte counts were determined. Picrosirius red-stained cervical tissue specimens were observed under a polarizing microscope, and the percentage of type I and type III collagen areas in the cervical tissue were calculated. The PMN% in cervical mucus was lowest at 200 days gestation (12–13 weeks before delivery), significantly increased 5 weeks before (21.7 ± 0.04), and was highest 1 week before calving (50.9 ± 0.04). IL-8 levels were increased at 295 days compared with those at 200 days of pregnancy (p < 0.05). No significant changes were observed in the white blood cell counts. The percentage of type I collagen in the cervical tissue reached a maximum (91.4 ± 0.02%) on day 200, significantly decreased after 274 days (3 weeks before calving), and continued to decrease thereafter until the week of parturition. There was no significant change in type III collagen levels. The results suggest that cervical ripening progresses when PMNs begin to infiltrate the cervix at around 260 days of gestation (5–4 weeks before parturition), IL-8, which increases at the end of pregnancy, mobilizes PMNs, and enhances inflammation, and that type I collagen changes are useful as an indicator of cervical ripening.