Collagen Fraction Research Articles

Investigation of the intrinsic permeability of ice-templated collagen scaffolds as a function of their structural and mechanical properties

Collagen scaffolds are widely used in a range of tissue engineering applications, both in vitro and in vivo, where their permeability to fluid flow greatly affects their mechanical and biological functionality. This paper reports new insights into the interrelationships between permeability, scaffold structure, fluid pressure and deformation in collagen scaffolds, focussing in particular on the degree of closure and the alignment of the pores.Isotropic and aligned scaffolds of different occlusivity were produced by ice templating, and were characterised in terms of their structure and mechanical properties. Permeability studies were conducted using two experimental set-ups to cover a wide range of applied fluid pressures. The permeability was found to be constant at low pressures for a given scaffold with more open structures and aligned structures being more permeable. The deformation of scaffolds under high pressure led to a decrease in permeability. The aligned structures were more responsive to deformation than their isotropic equivalents with their permeability falling more quickly at low strain. For isotropic samples, a broad (1 − ɛ)2 dependence for permeability was observed with the constant of proportionality varying with collagen fraction as the starting structures became more occluded. Aligned scaffolds did not follow the same behaviour, with the pores apparently closing more quickly in response to early deformation. These results highlight the importance of scaffold structure in determining permeability to interstitial fluid, and provide an understanding of scaffold behaviour within the complex mechanical environment of the body. Statement of significanceCollagen scaffolds are widely used in tissue engineering applications, for instance to contribute with wound healing. Their permeability to fluid flow, such as water and blood, is important to ensure they perform efficiently when inside the body. The present study reports new insights into the relationships between permeability, scaffold structure, fluid pressure and deformation in collagen scaffolds. It presents in particular the experimental setups used to measure these properties and the result of comparisons between collagen scaffolds with different structures: aligned and isotropic (non-aligned). It indicates quantitative differences in terms of permeability, and the effects of compression on such permeability. The results contribute to the development and understanding of collagen scaffolds and their applications.

Diet and Radiocarbon Dating of Tollund Man: New Analyses of an Iron Age Bog Body from Denmark

ABSTRACTTollund 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).

Dexmedetomidine restores autophagy and cardiac dysfunction in rats with streptozotocin-induced diabetes mellitus.

Dexmedetomidine (DEX), a highly selective and potent α2-adrenergic receptor agonist, has anti-apoptotic, anti-inflammatory, and anti-oxidative stress effects in diabetes mellitus (DM) rats. The underlying molecular mechanisms and signaling pathways of diabetic cardiomyopathy remain poorly understood. This study aimed to elucidate the effect of DEX on cardiac function in DM rats. Eight-week-old male Sprague Dawley rats were divided into three groups: control (n = 5), diabetes (DM, n = 7), and diabetes + DEX (DM + DEX, n = 10). DM was induced via intraperitoneal injection of streptozotocin (70mg/kg); at 3days later, DEX (1µg/kg/h) was administered for 4weeks. Cardiac function was evaluated using pressure-volume loop analysis and echocardiography. Left ventricular (LV) histological sections were used to analyze the interstitial collagen fraction. Using the LV samples, we performed a western blot analysis to evaluate signaling pathways and autophagic markers. The DM group had lower body weight and higher blood glucose level and heart weight/body weight ratio than the control group. However, metabolic changes did not differ between the DM and DM + DEX groups. Pressure-volume loop analysis and echocardiography showed impaired cardiac function, evidenced by a decrease in systolic and diastolic function, in both DM groups. DEX treatment in DM rats was associated with increased LV end-systolic pressure, LV contractility, cardiac output, and relaxed LV function compared with that in non-treated DM rats. LC3B and autophagy-related gene (ATG) proteins increased in the hearts of DM rats compared with the hearts of control rats. However, DEX reduced the expression of LC3B and ATG proteins in the hearts of DM rats. Increased p-ERK and decreased p-AKT were reduced in the hearts of DEX-treated DM rats. DEX reduces cardiac dysfunction and impaired autophagy in DM rats. This study reinforces our understanding of the potential anti-autophagic effect of DEX in patients with diabetic cardiomyopathy.

Exercise Hemodynamics After Aortic Valve Replacement for Severe Aortic Stenosis

Severe aortic stenosis (AS) is often accompanied by diastolic dysfunction. After aortic valve replacement (AVR), the left ventricle often undergoes considerable reverse remodeling. Despite this, diastolic dysfunction may persist after AVR. The aims of this study were to determine the incidence of elevated left ventricular (LV) filling pressure at rest and during exercise among patients with severe AS after AVR and to describe factors related to elevated LV filling pressure, especially its association with LV and left atrial remodeling and myocardial fibrosis. Thirty-seven patients undergoing AVR were included. Echocardiography, cardiac computed tomography, and magnetic resonance imaging were performed before AVR. An LV biopsy sample was obtained during AVR and analyzed for collagen fraction. One year after AVR, right heart catheterization with exercise was performed. A mean pulmonary capillary wedge pressure (PCWP)≥28mm Hg during exercise was considered elevated. Twelve patients (32%) had elevated exercise PCWP 1year after AVR. Exercise PCWP was highest among patients undergoing concomitant coronary artery bypass graft surgery (30±7 vs 25±6mm Hg, P=.04) and among patients with preoperative stroke volume index<35mL/m2 (28±8 vs 23±4mm Hg, P<.05). Baseline LV ejection fraction was lower among patients with elevated PCWP (56±8% vs 64±8%, P=.01), and coronary calcium score was significantly higher (median 870 AU [interquartile range, 454-2,491 AU] vs 179 AU [interquartile range, 63-513 AU], P=.02). Conversely, exercise PCWP was not related to the presence of high LV wall mass or to the severity of AS. Among patients undergoing isolated AVR, there was a correlation between LV interstitial volume fraction and PCWP (r=0.57, P=.01) and mean pulmonary artery pressure (r=0.51, P=.03) during exercise. Elevated filling pressure during exercise was seen in one third of patients after AVR in this population and was seen primarily among patients with coexisting ischemic heart disease or diffuse myocardial fibrosis but was unrelated to preoperative severity of AS and LV remodeling.

Doxycycline Attenuates Atrial Remodeling by Interfering with MicroRNA-21 and Downstream Phosphatase and Tensin Homolog (PTEN)/Phosphoinositide 3-Kinase (PI3K) Signaling Pathway.

BackgroundAtrial remodeling especially in the form of fibrosis is the most important substrate of atrial fibrillation (AF). The aim of this study was to investigate the effects of doxycycline on chronic intermittent hypoxia (CIH)-induced atrial remodeling and the pathophysiological mechanisms underlying such changes.Material/MethodsA total of 30 Sprague-Dawley rats were randomized into 3 groups: Control group, CIH group, and CIH with doxycycline treatment group. CIH rats were subjected to CIH 6 h/d for 30 days and treatment rats were administrated doxycycline while they received CIH. After the echocardiography examination, rats were sacrificed at 31 days. The tissues of atria were collected for histological and molecular biological experiments, Masson staining was used to evaluate the extent of atrial fibrosis, microRNA-21, and its downstream target phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K) were assessed.ResultsCompared to the control group, the CIH rats showed higher atrial interstitial collagen fraction, increased microRNA-21, PI3K levels, and decreased PTEN levels. Doxycycline treatment attenuated CIH-induced atrial fibrosis, reduced microRNA-21 and PI3K, and increased PTEN.ConclusionsCIH induced significant atrial remodeling, which was attenuated by doxycycline in our rat model. These changes may be explained due to alterations in the microRNA-21-related signaling pathways by doxycycline.

Biological response of human osteosarcoma cells to Si3N4-doped Bioglasses

In vitro and in vivo studies have recently shown that silicon nitride (Si3N4) is an osseoconductive and potential osseoinductive biomaterial. Even though the amount of bone mineral produced by Si3N4 was inferior when compared to commercial Bioglass®, it strongly stimulated the production of extracellular matrix and collagen tissues. Consequently, this study was designed to determine if appropriate mixtures of Bioglass® and Si3N4 might improve the ratio of mineral apatite to its biological matrix. Commercial 45S5 Bioglass® powder was mixed with 5 and 10 mol% Si3N4 powder, followed by melting, cooling, crushing, and comminution. The composite powders had modified morphologies and chemical structures including a reduced average particle size, a reduction of oxygen in the SiO backbone structure, and trapped CO2 gas which was converted into calcium carbonate. When exposed to human osteosarcoma cells within an osteogenic medium, the 5 mol% Si3N4 composite powder resulted in higher amounts of hydroxyapatite while the 10 mol% Si3N4 powder provided in a more balanced combination of collagen and mineral fractions closer to the natural composition of native human bone.

Short time insulin treatment post burn improves elastic-collagen rearrangement and reepithelization

ABSTRACTExtensive burn may cause acute resistance to insulin, which accentuates hypermetabolism, impairs glucose metabolism, immune dysfunction and risks of sepsis. To minimize these effects, insulin is used as a treatment. The purpose was to analyze the collagen-elastic arrangement effects of insulin on the burned skin. Wistar rats were assigned in groups: control (C); control with insulin (C + I); scald burn injury (SBI); and SBI with insulin (SBI+ I). SBI were submitted to 45% total body surface area burn and the insulin-treated groups received insulin (5 UI/Kg/day) for 4 or 14 days (d). Insulin levels, glucose tolerance test and HOMA index were determined. The skin sections were analyzed for histophatological and morphoquantitative data. Histopathological findings showed increased reepithelization of SBI+ I and formation of a new muscle layer after 14 days. In the collagen-elastic arrangement, insulin for 4 days increased the volume fraction (Vv) of thin collagen and elastic fibers. After 14 days, independently of injury, insulin decreased the elastic fibers. Insulin was able to reverse damages in the collagen-elastic rearrangement and stimulate reepithelization after 4 days. Untreated scald-burned animals showed higher Vv of thick collagen after 4 days, while those treated had a higher Vv of thin collagen. The Vv of elastic fibers was increased in SBI+ I for 4 days. In conclusion, insulin treatment was able to stimulate reepithelization. It also reversed the damages to the collagen-elastic arrangement in the scald-burned group, improving the organization of thin collagen and increasing the Vv of elastic fibers in the injured group treated with insulin for a short time, that is, for 4 days.

Guanacos y ecología isotópica en el norte del Neuquén: El registro de Cueva Huenul 1

En este trabajo se presentan los primeros resultados de ecología isotópica regional para el extremo norte de la provincia del Neuquén (Argentina). Esta información es clave para la reconstrucción de cambios ecológicos y paleodietas humanas a través del tiempo. Se exponen los resultados obtenidos a partir de la medición de isótopos estables de carbono (13C/12C) y nitrógeno (15N/14N) sobre el colágeno óseo de 39 muestras de fauna y una muestra humana procedentes de sitios arqueológicos de la localidad Barrancas-Buta Ranquil. Se presentan datos para cinco especies silvestres, aunque el foco del análisis se centra en guanaco (Lama guanicoe). La información es interpretada en el marco de los resultados obtenidos de muestreos de vegetación en la región, los cuales indican la distribución y abundancia de especies con las distintas vías fotosintéticas. Debido a su posición, adyacente al ecotono entre las comunidades fitogeográficas del Monte y la Patagonia, la región ofrece un contexto adecuado para el análisis de cambios ecológicos a través del tiempo. La totalidad de las especies vegetales relevadas en el muestreo sistemático siguen la vía fotosintética C3. En concordancia con esto, los valores de especies animales fueron relacionados a una cadena trófica C3. El entierro humano del sitio Buta Có Abajo registró valores que sugieren una dieta compuesta por guanacos y que además incluyó animales pequeños con valores más enriquecidos. En cuanto a los guanacos, se registraron diferencias isotópicas entre las muestras asignadas al Holoceno temprano y el tardío; estas últimas reflejan un mayor consumo de especies con vía fotosintética C3. Por último, en este conjunto de muestras, no se registró relación entre la altitud y los valores isotópicos de ?13C y ?15N, mientras que se observaría una tendencia latitudinal en la que los camélidos del centro sur de Mendoza presentan un mayor consumo de especies vegetales C4 que las procedentes del norte de Neuquén.

Protein Kinase D was involved in vascular remodeling in spontaneously hypertensive rats

ABSTRACTThe present study was designed to determine the role of PKD in vascular remodeling (VR) in Spontaneously hypertensive rats (SHRs). Increased SBP, VR and PKD activation were prominent in SHRs. The SBP has a positive correlation with the activation of PKD in SHRs. The ratio of media to lumen (MT/LD), volume fraction of collagen (VFC), hydroxyproline, IL-6, TNF-α and nitrotyrosine content were significantly related to the activated PKD. It may be concluded that PKD plays a central role in VR, and the mechanism may be related to its regulation of hypertrophy, fibrosis, inflammation and oxidative stress.

Measurement of protein and collagen in teeth before and after whitening strips

Americans spend billions of dollars each year on whitening products for the teeth. Whether it is performed in a dentist's office or with over‐the‐counter whitening strips, the active ingredient, hydrogen peroxide, is the same and has long been known to damage proteins. The goal was to measure the level of protein extracted from teeth untreated as well as those treated with one or three rounds of whitening strips. Teeth were purchased and treated according to manufacturer's instructions. In one round, teeth were dry when treated and washed with water after for 30 minutes. In another round, teeth were suspended in artificial saliva during treatment and for 30 minutes afterwards. Afterwards, the teeth were dialyzed against 10% EDTA pH 9.0 for 2 weeks. The fluid contained the soluble non collagen proteins and total protein was measured using the Lowry assay. The tooth remnants were dialyzed for two weeks against water and placed in liquid nitrogen, hammered in shards and then ground into a powder in a blender. The powder was suspended in an acetic acid, sodium chloride solution and contained collagen. The collagen was treated with pepsin for four hours to collect a pepsin soluble fraction (PSC). The amount of protein in the collagen and PSC fraction was tested using a modified Lowry assay procedure. Based on the Lowry assay, there was a significant decrease in protein content after treatment with whitening strips and the loss increased with more treatments. Based on the modified Lowry procedure, there was also a significant loss of collagen as measured by the modified Lowry procedure for both the collagen and PSC fraction. Similar losses were observed with or without the artificial saliva.Support or Funding InformationAuthors thank Stockton University for the Research and Professional Development grant to support this project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Extraction and Separation of Collagen on SDS PAGE to Determine Effect of Whitening Strips on Teeth

In the United States, billions of dollars are spent each year of tooth whitening products including treatments in dentist's offices as well as whitening strips which are sold over the counter. Both of these products use hydrogen peroxide as the active ingredient which has long been known to damage proteins. The outermost part of teeth is the enamel which contains several soluble proteins and the dentin underneath contains nearly 90 to 95 % collagen. The goal of this project was to extract collagen from both untreated teeth as well as those treated with whitening strips and examine the collagen on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Teeth were placed into artificial saliva and either treated with whitening strips for one or three rounds according to manufacturer's instructions or were untreated. Following a treatment, teeth were placed in artificial saliva for 30 minutes. Following treatments, teeth were dialyzed against 10% EDTA, pH 9.0 for two weeks with several changes of EDTA solution. The teeth were then dialyzed against water for two weeks with several changes. The tooth remnants were placed in liquid nitrogen, hammered into shards and ground into a powder using a blender. The powder was suspended in 0.5 M acetic acid and is the collagen fraction (COL); it was treated with pepsin for four hours, neutralized with TRIS, centrifuged and the supernatant was the pepsin soluble collagen fraction (PSC). Gels of varying concentrations of acrylamide were prepared and COL and PSC fractions were loaded. Two modified procedures for the Coomassie Blue staining protocol were used. In one, the washing solution did not contain methanol and this caused the COL proteins to turn a pink color. In addition, the gels were stained with the Oriole fluorescent stain. In order to determine the type of collagen present, standards made from collagen I and III were also used. Results will be presented that shows the level of COL in treated and untreated teeth.Support or Funding InformationFunding was provided by the Research and Professional Development Grant from Stockton University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Using Hydroxyproline to Measure Collagen in Teeth With and Without Whitening Strips

Billions of dollars are spent in the United States every year on tooth whitening. Whether it is done in a dentist's office or with over the counter products, the active ingredient is often hydrogen peroxide which has long been known to damage proteins. The outermost part of the tooth is the enamel and contains several proteins while dentin, the layer underneath, contains 90 to 95% collagen. This protein contains the modified amino acid hydroxyproline and the level of this amino acid has been used to measure the amount of collagen. Hydroxyproline can be detected using a colorimetric test: the original test required use of perchloric acid but the method was modified with use of hydrochloric acid replacing the perchloric acid. Teeth were purchased, placed in artificial saliva and treated with whitening strips following the manufacturer's instructions while other teeth did not receive the strips. Afterwards, the teeth were dialyzed against 10% EDTA, pH 9.0 for 2 weeks to remove calcium and then dialyzed against water to wash the teeth. They were then placed in liquid nitrogen and hammered into shards which were turned into a powder using a blender. The powder was suspended in an acetic acid solution to make the collagen fraction(COL). This fraction was treated with pepsin for four hours, neutralized with saturated Tris solution and centrifuged for 1 hour. The supernatant was the pepsin soluble fraction (PSC). Both COL and PSC were then hydrolyzed by adding 6 M HCl overnight at 95 °C. The resulting hydrolyzed samples were neutralized with 10 M sodium hydroxide and tested for hydroxproline. Results indicated a significant drop in hydroxproline content after one treatment and an even greater loss after three treatments. The drop in the level of hydroxyproline following treatment with whitening strips does suggest a loss of collagen from the dentin due to the whitening strips.Support or Funding InformationAuthors express gratitude to Research and Professional Development Grant from Stockton University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Overexpression of prostaglandin E2 EP4 receptor improves cardiac function after myocardial infarction

BackgroundProstaglandin E2 (PGE2) signals through 4 separate G-protein coupled receptor sub-types to elicit a variety of physiologic and pathophysiological effects. We recently reported that PGE2 via its EP3 receptor could reduce cardiac contractility of isolated myocytes and the working heart preparation. We thus hypothesized that there is an imbalance in the EP3/EP4 ratio towards EP3 in the failing heart and that overexpression of EP4 in a mouse model of heart failure would improve cardiac function. Methods and resultsOur hypothesis was tested in a mouse model of myocardial infarction (MI) with the use of AAV9-EP4 driven by the myosin heavy chain promoter to overexpress EP4 in the cardiac myocytes. Echocardiography was performed to assess cardiac function. We found that overexpression of EP4 improved shortening fraction (p = 0.0025), ejection fraction (p = 0.0003), and reduced left ventricular dimension at systole (p = 0.0013). Overexpression of EP4 also significantly reduced indices of cardiac hypertrophy and interstitial collagen fraction. Animals treated with AAV9-EP4 also had a significant decrease in TNFα mRNA expression and in the number of macrophages and T cells migrated post MI coupled with a reduction in the expression of iNOS. ConclusionOverexpression of EP4 improves cardiac function post MI. This may be mediated through reductions in adverse cardiac remodeling or via inhibition of cytokine/chemokine production.

Doxycycline attenuates chronic intermittent hypoxia-induced atrial fibrosis in rats.

Atrial structural remodeling in the form of fibrosis contributes to the arrhythmic substrate in atrial fibrillation (AF). The aim of this study was to investigate the effects of doxycycline on chronic intermittent hypoxia (CIH)-induced atrial fibrosis and the pathophysiological mechanisms underlying such changes. A total of 30 Sprague Dawley rats were randomized into three groups: control group, CIH group, and CIH with doxycycline treatment (CIH-D) group. CIH lasted 5hours per day for 4weeks. CIH-D rats were administrated doxycycline for 4weeks, while they received CIH. Masson's trichrome staining was used to determine collagen deposit in the atrial myocardium. Protein and mRNA levels of Matrix Metalloproteinase-2 (MMP-2) and -9 (MMP-9), microRNA-21 (miR-21) and its downstream target Sprouty1 (Spry1), and extracellular signal-regulated kinases 1/2 (ERK1/2) were measured using Western blotting or real-time qRT-PCR, respectively. Compared to the control group, the CIH group showed higher interstitial collagen fraction, increased MMP-9, miR-21, and p-ERK1/2 levels, and decreased MMP-2 and Spry1 levels. Doxycycline treatment attenuated CIH-induced atrial fibrosis, reduced MMP-2, MMP-9, miR-21, and p-ERK1/2, and increased Spry1. CIH treatment induced significant atrial fibrosis in our rat model, which was attenuated by doxycycline. These changes can be explained by alterations in the MMP and miR-21/ERK signaling pathways.

Cross-Linked Cholecyst-Derived Extracellular Matrix for Abdominal Wall Repair.

Abdominal wall repair frequently utilizes either nondegradable or biodegradable meshes, which are found to stimulate undesirable biological tissue responses or which possess suboptimal degradation rate. In this study, a biologic mesh prototype made from carbodiimide cross-linked cholecyst-derived extracellular matrix (EDCxCEM) was compared with small intestinal submucosa (Surgisis®), cross-linked bovine pericardium (Peri-Guard®), and polypropylene (Prolene®) meshes in an in vivo rabbit model. The macroscopic appearance and stereological parameters of the meshes were evaluated. Tailoring the degradation of the EDCxCEM mesh prevents untimely degradation, while allowing cellular infiltration and mesh remodeling to take place in a slower but predictable manner. The results suggest that the cross-linked biodegradable cholecyst-derived biologic mesh results in no seroma formation, low adhesion, and moderate stretching of the mesh. In contrast to Surgisis, Peri-Guard, and Prolene meshes, the EDCxCEM mesh showed a statistically significant increase in the volume fraction (Vv) of collagen (from 34% to 52.1%) in the central fibrous tissue region at both day 28 and 56. The statistically high length density (Lv), of blood vessels for the EDCxCEM mesh at 28 days was reflected also by the higher cellular activity (high Vv of fibroblast and moderate Vv of nuclei) indicating remodeling of this region in the vicinity of a slowly degrading EDCxCEM mesh. The lack of mesh area stretching/shrinkage in the EDCxCEM mesh showed that the remodeled tissue was adequate to prevent hernia formation. The stereo-histological assays suggest that the EDCxCEM delayed degradation profile supports host wound healing processes including collagen formation, cellular infiltration, and angiogenesis. The use of cross-linked CEM for abdominal wall repair is promising.

The Turning Point for Morphomechanical Remodeling During Complete Intestinal Obstruction in Rats Occurs After 12-24h.

Intestinal obstruction prompts luminal dilation and wall remodeling proximal to the site of obstruction. Studies on temporal and spatial morphomechanical remodeling are needed for comprehending the pathophysiology of acute intestinal obstruction. The aim was to estimate the no-load and zero-stress morphomechanical properties in circumferential and longitudinal direction at 0, 6, 12, 24, 36, and 48h after complete intestinal obstruction. Obstruction of the distal ileum was created surgically by placement of a polyethylene ring for up to 48h in 30 rats. Sham and normal groups were also studied (n=12). Five 6cm-long intestinal segments proximal to the obstruction site were used for histological, morphometric and mechanical analysis at the designated times. Morphomechanical changes were huge but only subtle changes were observed between the 5 segments during the obstruction period. Due to dilation, the serosal length and mucosal length increased continuously from 6 to 48h (p<0.001). The wall area increased at 24h and beyond (p<0.001), demonstrating tissue growth. The opening and bending angle decreased to minimum values at 24h where after the opening angle increased and the bending angle returned to pre-obstruction levels. For the residual stretch ratios and the position of the neutral axis the turning point was found after 24h. Histologically, the thickness and area of most wall layers were quite stable for the first 12h but with an increase at the 24h time point that continued to the 48h time point. The most pronounced change was found for the circumferential muscle layer (p<0.05). Analysis of picrosirius red stained slides showed that submucosal type 3 collagen fraction increased significantly (p<0.001), whereas the fraction of type 1 collagen decreased (p<0.001). In conclusion, pronounced time-dependent morphomechanical remodeling was found. The obstructed intestine went from dilation remodeling to growth remodeling during the interval 12-24h after creating the obstruction.

Spectroscopic imaging: Nuclear magnetic resonance and Raman spectrometry for the detection of collagen cross-linking from giant squid mantle, fin, and tentacle tissues

ABSTRACTThe application of proton nuclear magnetic resonance (1H-NMR) imaging and Raman spectrometry is described for the detection and structural characterization of collagen cross-linking (pyridinoline) in soluble and insoluble collagen fractions obtained by cation-exchange separation of the mantle, fins, and tentacles of giant squid (Dosidicus gigas). The pyridinoline was detected using a fluorometric procedure only in the soluble collagen fractions. However, the pyridinoline was detected using high-performance liquid chromatography (HPLC) only in the insoluble collagen fraction from the tentacles. Meanwhile, in all samples, pyridinoline was detected by 1H-NMR and Raman spectrometry. The higher peak intensities in both methods were observed in the insoluble collagen fraction from the tentacles. The muscles showed a lower decrease in shear force after 30 min of cooking. 1H-NMR and Raman spectrometry provides an assay which is more sensitive than fluorescence and HPLC for pyridinoline detection in squid tissues, suggesting greater potential for the analysis of different types of tissue and collagen fractions.

Influence of apocynin on cardiac remodeling in rats with streptozotocin-induced diabetes mellitus

BackgroundIncreased reactive oxygen species (ROS) generation in diabetes mellitus (DM) is an important mechanism leading to diabetic cardiomyopathy. Apocynin, a drug isolated from the herb Picrorhiza kurroa, is considered an antioxidant agent by inhibiting NADPH oxidase activity and improving ROS scavenging. This study analyzed the influence of apocynin on cardiac remodeling in diabetic rats.MethodsSix-month-old male Wistar rats were assigned into 4 groups: control (CTL, n = 15), control + apocynin (CTL + APO, n = 20), diabetes (DM, n = 20), and diabetes + apocynin (DM + APO, n = 20). DM was induced by streptozotocin. Seven days later, apocynin (16 mg/kg/day) or vehicle was initiated and maintained for 8 weeks. Left ventricular (LV) histological sections were used to analyze interstitial collagen fraction. NADPH oxidase activity was evaluated in LV samples. Comparisons between groups were performed by ANOVA for a 2 × 2 factorial design followed by the Bonferroni post hoc test.ResultsBody weight (BW) was lower and glycemia higher in diabetic animals. Echocardiogram showed increased left atrial diameter, LV diastolic diameter, and LV mass indexed by BW in both diabetic groups; apocynin did not affect these indices. LV systolic function was impaired in DM groups and unchanged by apocynin. Isovolumic relaxation time was increased in DM groups; transmitral E/A ratio was higher in DM + APO compared to DM. Myocardial functional evaluation through papillary muscle preparations showed impaired contractile and relaxation function in both DM groups at baseline conditions. After positive inotropic stimulation, developed tension (DT) was lower in DM than CTL. In DM + APO, DT had values between those in DM and CTL + APO and did not significantly differ from either group. Myocardial interstitial collagen fraction was higher in DM than CTL and did not differ between DM + APO and CTL + APO. Serum activity of antioxidant enzymes glutathione peroxidase, superoxide dismutase (SOD), and catalase was lower in DM than CTL; apocynin restored catalase and SOD levels in DM + APO. Myocardial NADPH oxidase activity did not differ between groups.ConclusionApocynin restores serum antioxidant enzyme activity despite unchanged myocardial NADPH oxidase activity in diabetic rats.

Physicochemical changes of pepsin-solubilized and insoluble collagen in jumbo squid (Dosidicus gigas) muscle after cooking process

ABSTRACTCollagen is the major connective tissue (CT) protein and one of the main constituents of the jumbo squid (Dosidicus gigas). Therefore, physicochemical changes of pepsin-solubilized collagen (PSC) and insoluble collagen (IC) were studied after cooking (100°C/30 min) of muscle (mantle, fins, and arms). Different pyridinoline (Pyr) contents (the major cross-linking molecule in collagen fibers) were found in the fresh muscle of the three anatomical regions. After the cooking process, a decrease from 10 to 30% in the thermal resistance of collagen was observed, depending on the anatomical region and fraction evaluated. Furthermore, the electrophoretic profile, Fourier transform infrared (FTIR) spectroscopy, and the amino-acid profile revealed that structural changes occurred in the two different collagen fractions caused by the thermal process, and the changes were greater in the mantle. Under the conditions applied in this study, collagen fractions from the squid arms showed more stability during the cooking process due to the high cross-linking degree of their fibers.

FIBROTIC CHANGES IN PATIENTS WITH CHRONIC HEART FAILURE WITH CARDIAC DYSSYNCHRONY AND ASSOCIATED TYPE 2 DIABETES MELLITUS

The study of fibrosis markers was carried out on 72 observed patients (mean age (69 ± 10.37) years) with chronic heart failure (CHF) of ischemic genesis with manifestations of cardiac dyssynchrony (CD) and concomitant type 2 diabetes mellitus – Galectin 3 and matrix metalloproteinase 1. All patients were divided into 2 groups, depending on the presence of CD. The CD was evaluated according to a conventional technique, the volume fraction of interstitial collagen was measured using the formula of J. Shirani and co-authors, the levels of Galectin-3 and matrix metalloproteinase 1 – by the enzyme-linked immunoassay according to the manufacturer's instructions. The data were processed using parametric and nonparametric statistics. It was revealed that the level of fibrosis development was higher in the group of patients with CD than in the group without CD. This indicates the dependence of the development of myocardial sites asynchronous reduction with the presence of interstitial collagen development. That further requires the study of the effect of anti-fibrotic, anti-ischemic and hypoglycemic agents on the progression of CD to prevent subsequent myocardial remodeling.