Total Collagen Content Research Articles

A non-invasive approach to assess texture changes in sous-vide cooked Atlantic mackerel during chilled storage by fluorescence imaging

The aim of this study was to investigate the potential of fluorescence microscopy coupled with chemical, physical and data analysis methods for reliable and non-invasive detection of changes in texture parameters of sous-vide cooked Atlantic mackerel during chilled storage. Fluorescence micrographs of cook loss and connective tissue of the fish samples after sous-vide treatment at 60 °C and 75 °C for 10, 15 and 20 min taken in the 1st, 3rd and 7th day of chilled storage were acquired. The obtained images were numerically processed and the resulting data was directly correlated (R = 0.960) with the total collagen content determined by a chemical method. Partial least squares analysis was applied to derive statistically significant regression models revealing the influence of each of sous-vide regime parameters on changes in total collagen content and texture parameters of Atlantic mackerel during chilled storage. Results showed that both collagen integrity and firmness of mackerel flesh were significantly (p < 0.05) affected by the temperature of sous-vide treatment and duration of chilled storage, leading to gradual softening of the fish tissue due to degradation of collagenous tissue.

The effects of hydroxyapatite nanoparticles embedded in a MMP-sensitive photoclickable PEG hydrogel on encapsulated MC3T3-E1 pre-osteoblasts

This study investigated the effects of introducing hydroxyapatite nanoparticles into a matrix metalloproteinase (MMP) sensitive poly(ethylene glycol) (PEG) hydrogel containing cell adhesion peptides of RGD for bone tissue engineering. MC3T3-E1 pre-osteoblasts were encapsulated in the biomimetic PEG hydrogel, which was formed from the photoclick thiol-norbornene reaction system, cultured for up to 28 d in growth medium or osteogenic differentiation medium, and evaluated by cellular morphology and differentiation by alkaline phosphatase (ALP) activity and bone-like extracellular matrix deposition for mineral and collagen. Hydroxyapatite nanoparticles were incorporated during hydrogel formation and cell encapsulation at 0%, 0.1% or 1% (w/w). Incorporation of hydroxyapatite nanoparticles did not affect the hydrogel properties as measured by compressive modulus and equilibrium swelling. In growth medium, encapsulated MC3T3-E1 cells remained largely round regardless of hydroxyapatite concentration. ALP activity increased by 25% at day 14 and total collagen content increased by 55% at day 28 with increasing hydroxyapatite concentration from 0% to 1%. In differentiation medium, cell spreading was evident regardless of hydroxyapatite indicating that the MC3T3-E1 cells were able to degrade the hydrogel. For the 1% hydroxyapatite condition, ALP activity was 27% higher at day 14 and total collagen content was 22% higher at day 28 in differentiation medium when compared to growth medium. Mineral deposits were more abundant and spatial elaboration of collagen type I was more evident in the 1% (w/w) hydroxyapatite condition with differentiation medium when compared to all other conditions. Overall, osteogenesis was observed in the hydrogels with hydroxyapatite nanoparticles in growth medium but was enhanced in differentiation medium. In summary, a biomimetic hydrogel comprised of MMP-sensitive crosslinks, RGD cell adhesion peptides, and 1% (w/w) hydroxyapatite nanoparticles is promising for bone tissue engineering.

Abstract 599: Exposure to Biodiesel Exhaust Triggers Atherosclerotic Plaque Destabilisation through Increased Apoptosis of Vascular Smooth Muscle Cells in the Arterial Vessel Wall

Introduction: Long-term exposure to air pollutants increases cardiovascular morbidity and mortality. Particulate matter (PM) derived from diesel exhaust has been documented to be pro-atherogenic in animal studies. Biodiesels are widely introduced as new fuels with improved emission characteristics. However, biodiesel contains relatively more polycyclic aromatic carbohydrates (PAH) compared to diesel. Therefore, we hypothesise that exposure to biodiesel as compared to diesel exhaust results in increased atherosclerosis. Methods and Results: In a carotid cuff atherosclerosis model, the effects of exposure to exhaust PM of biodiesel vs. diesel PM vs. saline (control) on atherosclerosis were evaluated. Both common carotid arteries in LDLR -/- mice were cuffed at week 2 in the course of an 8-week high-fat diet regimen. All mice were intratracheally instilled with saline, PM biodiesel or biodiesel once-weekly for 5 times. Immunohistochemistry and primary human vascular smooth muscle cells (hVSMC) were used for evaluation. Results: Exposure to both biodiesel and diesel didn’t exacerbate atherosclerosis development, however, biodiesel affected plaque composition towards a more vulnerable phenotype as compared to diesel with decreased total collagen content and hVSMC (respectively -100%, p=0.09 and -339%, p&amp;lt;0.01 and tend to increase necrotic core volumes (37.5% vs 28.9 % p=0.2). Exposure to biodiesel PM triggered loss of hVSMC in tunica-media, strongly correlating with apoptosis in the vessel wall (Pearson r=0.7, p&amp;lt;0.01). These findings were supported by dose-dependent apoptosis of hVSMCs upon 2-hour PAH treatment. Conclusions: This study demonstrates that exposure to biodiesel exhaust PM dramatically modulates atherosclerotic plaque composition, resulting in an unstable plaque phenotype through enhanced pro-apoptotic mechanisms.

Elevated urinary excretion of free pyridinoline in Friesian horses suggests a breed-specific increase in collagen degradation

BackgroundFriesian horses are known for their high inbreeding rate resulting in several genetic diseases such as hydrocephaly and dwarfism. This last decade, several studies focused on two other presumed hereditary traits in Friesian horses: megaoesophagus and aortic rupture. The pathogenesis of these diseases remains obscure but an important role of collagen has been hypothesized. The purpose of this study was to examine possible breed-related differences in collagen catabolism. Urinary specimens from Friesian (n = 17, median age 10 years old) and Warmblood horses (n = 17, median age 10 years old) were assessed for mature collagen cross-links, i.e. pyridinoline (PYD) (=hydroxylysylpyridinoline/HP) and deoxypyridinoline (DPD) (lysylpyridinoline /LP).Solid-phase extraction was performed, followed by reversed-phase ion-paired liquid chromatography prior to tandem mass spectrometry (MS/MS) detection.ResultsMean urinary concentrations of free PYD, expressed as fPYD/creatinine ratio, were significantly higher in Friesian horses compared to Warmblood horses (28.5 ± 5.2 versus 22.2 ± 9.6 nmol/mmol, p = 0.02) while mean fDPD/creatinine ratios were similar in both horse breeds (3.0 ± 0.7 versus 4.6 ± 3.7 nmol/mmol, p = 0.09).ConclusionsSince DPD is considered a specific bone degradation marker and PYD is more widely distributed in connective tissues, the significant elevation in the mean PYD/DPD ratio in Friesian versus Warmblood horses (9.6 ± 1.6 versus 5.7 ± 1.8, p < 0.0001) suggests a soft tissue origin for the increased fPYD levels. Considering that a previous study found no differences in total collagen content between Friesian and Warmblood horses for tendon and aortic tissue, this indicates a higher rate of collagen degradation. The latter might, at least in part, explain the predisposition of Friesians to connective tissue disorders.

Men are from Mars, Women are from Venus. Gender specific changes in pulmonary function in 1 year old rats after neonatal hyperoxia exposure

RationalePreterm birth affects about 11% of pregnancies worldwide. The Institute of Medicine estimated the cost associated with premature birth to be $36 billion in 2016. Lungs in preterm infants are in the critical saccular stage of lung development and susceptible to the oxygen‐induced chronic lung disease known as bronchopulmonary dysplasia (BPD). BPD is characterized by functional abnormalities including decreased saccular/alveolar septation, decreased microvascular cross‐sectional area with or without pulmonary hypertension, airway injury with or without increased airway reactivity, and abnormalities of control of breathing in various combinations. While the initial inflammation improves, chronic inflammation ensues for a long time. We tested how neonatal hyperoxia exposure affects lung function one year after birth. We postulated an increase in lung collagen levels reflected by reduced pulmonary function.Methods6 newborn male and female rats in each group were exposed to Hyperoxia (Hx; 85% O2) or Normoxia (Nx; 21% O2) for 14 days, then aged to 1 year in Nx. At one year, lung function was measured using a FlexiVent (Scireq, Montreal, Canada). Using a single frequency forced oscillation manoeuvre and a low‐frequency forced oscillation technique the lung was tested for compliance and resistance. Total lung collagen was determined by a colorimetric assay using Ehrlich's reagent.ResultsAnalysis of one year old rats showed no difference in weight and body length between the Nx and the Hx group within each gender. The inspiratory capacity (IC) was significantly higher in the Hx exposed male rats compared to the Nx exposed male rats (12.6±0.6 ml vs 14.0±0.9 ml; p=0.02). The difference in IC in female rats was significantly lower in the Hx group compared to the Nx group (11.3±0.4 vs 10.1±0.6 ml; p=0.002). Dynamic compliance was significantly increased in the male Hx group compared to the male Nx group (1.43±0.05 vs 1.61±0.11 ml/cm H2O; p=0.04). In contrast, the female Hx group showed significantly reduced compliance compared to the female Nx group (1.32±0.05 vs 1.15±0.17 ml/cm H2O). In addition, the PV loop area was higher in the male Hx group compared to the male Nx group and no difference was found between the two female groups. Total collagen in lung tissue was not different between the male Hx and Nx groups but was higher in the female Hx compared to the Nx group.ConclusionOne year after neonatal Hx exposure, the male lung shows increased compliance and inspiratory capacity compared to the Nx exposed rats suggesting a trend to emphysema. In contrast, female rats show signs of lung fibrosis reflected by reduced compliance and increased total collagen content. This data suggests that neonatal oxygen exposure initiates alterations in inflammatory and repair pathways that are different between male and female rats.Support or Funding InformationNHLBI: R01 HL115061‐01 (Eldridge)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Type III collagen affects dermal and vascular collagen fibrillogenesis and tissue integrity in a mutant Col3a1 transgenic mouse model

Type III collagen is a major fibrillar collagen consisting of three identical α1(III)-chains that is particularly present in tissues exhibiting elastic properties, such as the skin and the arterial wall. Heterozygous mutations in the COL3A1 gene result in vascular Ehlers-Danlos syndrome (vEDS), a severe, life-threatening disorder, characterized by thin, translucent skin and propensity to arterial, intestinal and uterine rupture. Most human vEDS cases result from a missense mutation substituting a crucial glycine residue in the triple helical domain of the α1(III)-chains. The mechanisms by which these mutant type III collagen molecules cause dermal and vascular fragility are not well understood. We generated a transgenic mouse line expressing mutant type III collagen, containing a typical helical glycine substitution (p.(Gly182Ser)). This Col3a1Tg-G182S mouse line displays a phenotype recapitulating characteristics of human vEDS patients with signs of dermal and vascular fragility. The Col3a1Tg-G182S mice develop severe transdermal skin wounds, resulting in early demise at 13–14weeks of age. We found that this phenotype was associated with a reduced total collagen content and an abnormal collagen III:I ratio, leading to the production of severely malformed collagen fibrils in the extracellular matrix of dermal and arterial tissues. These results indicate that expression of the glycine substitution in the α1(III)-chain disturbs formation of heterotypic type III:I collagen fibrils, and thereby demonstrate a key role for type III collagen in collagen fibrillogenesis in dermal and arterial tissues.

Devising tissue ingrowth metrics: a contribution to the computational characterization of engineered soft tissue healing

The paradigm shift brought about by the expansion of tissue engineering and regenerative medicine away from the use of biomaterials, currently questions the value of histopathologic methods in the evaluation of biological changes. To date, the available tools of evaluation are not fully consistent and satisfactory for these advanced therapies. We have developed a new, simple and inexpensive quantitative digital approach that provides key metrics for structural and compositional characterization of the regenerated tissues. For example, metrics provide the tissue ingrowth rate (TIR) which integrates two separate indicators; the cell ingrowth rate (CIR) and the total collagen content (TCC) as featured in the equation, TIR% = CIR% + TCC%. Moreover a subset of quantitative indicators describing the directional organization of the collagen (relating structure and mechanical function of tissues), the ratio of collagen I to collagen III (remodeling quality) and the optical anisotropy property of the collagen (maturity indicator) was automatically assessed as well. Using an image analyzer, all metrics were extracted from only two serial sections stained with either Feulgen & Rossenbeck (cell specific) or Picrosirius Red F3BA (collagen specific). To validate this new procedure, three-dimensional (3D) scaffolds were intraperitoneally implanted in healthy and in diabetic rats. It was hypothesized that quantitatively, the healing tissue would be significantly delayed and of poor quality in diabetic rats in comparison to healthy rats. In addition, a chemically modified 3D scaffold was similarly implanted in a third group of healthy rats with the assumption that modulation of the ingrown tissue would be quantitatively present in comparison to the 3D scaffold-healthy group. After 21 days of implantation, both hypotheses were verified by use of this novel computerized approach. When the two methods were run in parallel, the quantitative results revealed fine details and differences not detected by the semi-quantitative assessment, demonstrating the importance of quantitative analysis in the performance evaluation of soft tissue healing. This automated and supervised method reduced operator dependency and proved to be simple, sensitive, cost-effective and time-effective. It supports objective therapeutic comparisons and helps to elucidate regeneration and the dynamics of a functional tissue.

Changes in extracellular matrix in subcutaneous small resistance arteries of patients with essential hypertension

Background: In the development of hypertensive microvascular remodeling, a relevant role may be played by changes in extracellular matrix proteins. Aim of this study was the to evaluate some extracellular matrix components within the tunica media of subcutaneous small arteries in 9 normotensive subjects and 12 essential hypertensive patients, submitted to a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region.Patients and Methods: Subcutaneous small resistance arteries were dissected and mounted on an isometric myograph, and the tunica media to internal lumen ratio was measured. In addition, fibronectin, laminin, transforming growth factor-beta-1 (TGF-β1) and emilin-1 contents within the tunica media were evaluated by immunofluorescence and relative immunomorphometrical analysis (immunopositivity % of area). The total collagen content and collagen subtypes within the tunica media were evaluated using both Sirius red staining (under polarized light) and immunofluorescence assay.Results: Normotensive controls had less total and type III collagen in respect with hypertensive patients. Fibronectin and TGF-β1 tunica media content was significantly greater in essential hypertensive patients, compared with normotensive controls, while laminin and emilin-1 tunica media content was lesser in essential hypertensive patients, compared with normotensive controls. A significant correlation was observed between fibronectin tunica media content and media to lumen ratio.Conclusions: Our results indicate that, in small resistance arteries of patients with essential hypertension, a relevant fibrosis may be detected; fibronectin and TGF-β1 tunica media content is increased, while laminin and emilin-1 content is decreased; these changes might be involved in the development of small resistance artery remodeling in humans.

Mesenchymal stem cells inhibit hypoxia-induced inflammatory and fibrotic pathways in bladder smooth muscle cells.

Partial bladder outlet obstruction is a multifactorial urological condition in which hypoxia plays a significant role. We recently investigated hypoxia's role as a single stressor and found that hypoxia induced an intense inflammatory and profibrotic switch in bladder smooth muscle cells (bSMCs). With the immunomodulatory capacity of mesenchymal stem cells (MSCs), we aimed to investigate if the hypoxia-signaling pathways can be mitigated using MSCs. Bladder smooth muscle cells were cultured in 3% oxygen tension for 72h with either the direct or indirect co-culture with bone marrow derived MSCs. High pore density transwells were used for indirect co-cultures. Total RNA was extracted for gene expression analysis and the Mesoscale multiplex assay was used for secreted cytokines and growth factor measurements. Total collagen contents were determined using the Sirius Red collagen assay. Hypoxia induced increase of HIF3α, VEGF, TGFβ1, TNFα, IL-1β, IL-6, αSMA, and total collagen expression and decreased IL-10 levels in bSMCs. Both direct and indirect MSCs co-cultures inhibited>50% of hypoxia-induced TGFβ1 and IL-6 expression (p<0.005) in a HIF-independent manner. Also, both MSCs co-culture techniques induced>200% increase in IL-10 protein (p<0.005) and inhibited hypoxia-induced αSMA, collagen I and III transcripts as well as total collagen proteins (p<0.0001). Contrastingly, the hypoxia-induced IL-1β and TNFα were inhibited by only the direct co-cultures (p<0.05). MSCs co-culture with bSMCs potently mitigates hypoxia-induced inflammatory and profibrotic pathways. This work has elucidated the role of cell-cell contact and paracrine immunomodulatory mechanisms of MSCs action and opened avenues for therapeutic intervention.

Determination of selenium during pathogenesis of hepatic fibrosis employing hydride generation and inductively coupled plasma mass spectrometry.

Serum and liver selenium levels were studied during the pathogenesis of N-nitrosodimethylamine (NDMA) induced hepatic fibrosis in rats. The degree of fibrosis was assessed with Masson's trichrome staining and quantifying collagen content in the liver. Lipid peroxides were measured in blood and liver samples and total glutathione and glutathione peroxidase were assayed in the liver tissue to evaluate oxidative stress. Interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β1) were measured in the serum. Selenium levels were determined using inductively coupled plasma-mass spectrometry (ICP-MS) after acid digestion and hydride generation of selenium. Serial administrations of NDMA produced well-developed fibrosis and early cirrhosis in the liver with 4-fold increase of total collagen content and deposition of collagen fibers. Blood and hepatic lipid peroxides, serum IL-6 and TGF-β1 were significantly increased. There was significant reduction in hepatic glutathione and glutathione peroxidase levels. Serum and liver selenium were remarkably decreased on all the days studied. The results suggest that decreased selenium and glutathione peroxidase contribute to the impairment of cellular antioxidant defense, which in turn results in oxidative stress and trigger pathogenesis of hepatic fibrosis. The study further demonstrated that ICP-MS with hydride generation technique is a reliable and sensitive method for determination of selenium in biological samples.

Effect of Melatonin on the Renin-Angiotensin-Aldosterone System in l-NAME-Induced Hypertension.

The renin-angiotensin-aldosterone system (RAAS) is a dominant player in several cardiovascular pathologies. This study investigated whether alterations induced by l-NAME, (NLG)-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor, and the protective effect of melatonin are associated with changes in the RAAS. Four groups of 3-month-old male Wistar rats (n = 10) were treated as follows for four weeks: untreated controls, rats treated with melatonin (10 mg/kg/day), rats treated with l-NAME (40 mg/kg/day), and rats treated with l-NAME + melatonin. l-NAME administration led to hypertension and left ventricular (LV) fibrosis in terms of enhancement of soluble, insoluble and total collagen concentration and content. Melatonin reduced systolic blood pressure enhancement and lowered the concentration and content of insoluble and total collagen in the LV. The serum concentration of angiotensin (Ang) 1–8 (Ang II) and its downstream metabolites were reduced in the l-NAME group and remained unaltered by melatonin. The serum aldosterone level and its ratio to Ang II (AA2-ratio) were increased in the l-NAME group without being modified by melatonin. We conclude that l-NAME-hypertension is associated with reduced level of Ang II and its downstream metabolites and increased aldosterone concentration and AA2-ratio. Melatonin exerts its protective effect in l-NAME-induced hypertension without affecting RAAS.

Taurine supplementation prevents endothelial dysfunction and attenuates structural changes in aortas from hypothalamic obese rats.

Obesity predisposes to cardiovascular and metabolic diseases. The amino acid, L-taurine (Tau), regulates glucose and lipid homeostasis and vascular function. Here we investigated whether Tau supplementation prevents endothelial dysfunction in the thoracic aortas of monosodium glutamate-induced obese (MSG) rats. Male rats received subcutaneous injections of MSG (4mg/kg body weight/day) or saline (control group, CTL) during the first five days of life. From 21 to 150days of age, the rats were distributed into the groups: CTL, MSG, and CTL and MSG supplemented with 2.5% Tau in their drinking water (CTAU and MTAU). At 150-days old, MSG rats presented massive abdominal fat deposition, hypertriglyceridemia, hyperinsulinemia, glucose intolerance and high plasma levels of malondialdehyde (MDA), a lipid peroxidation marker. Tau supplementation attenuated fat accumulation in perigonadal adipose tissue and prevented the increase in triglycerides and MDA plasma levels. Aortic rings of MSG rats presented reduced vasodilation in response to acetylcholine (ACh). No modifications in insulin-induced vasodilatation, or Akt and eNOS phosphorylation, were observed in MSG aortas; thoracic aortas from MSG rats presented reduced tunica media thickness, with a lower aortic wall thickness/lumen diameter ratio and decreased total collagen content. Tau supplementation restored ACh-induced vasodilation and collagen content. Our study presents the first evidence that Tau prevents disruptions in vascular reactivity and in extracellular matrix composition in thoracic aortas of MSG-obese rats. The vascular protective actions of Tau may be linked to reduced lipid peroxidation and a reduction in cardiovascular risk factors, such as abdominal fat and hypertriglyceridemia.

The effect of water vapor pressure on muscle collagen solubility and selected characteristics of the longissimus lumborum muscle in crossbred cattle

Abstract Most scientific studies are dedicated to the possibility of preparing beef for consumption under industrial conditions. Few publications are devoted to the issue of collagen thermohydrolysis in conditions available to the consumer. This study has analyzed the effect of small values of water vapor pressure on major culinary indices and chemical components of the longissimus lumborum muscle obtained from bulls with different growth rates. The experiment involved 48 animals. On the basis of the gain during the fattening time, the animals were divided into a low growth intensity group, with a daily body weight gain of ≤900 g, and a high growth intensity group with a daily gain of &gt;900 g/day. A part of the samples of the longissimus lumborum muscle (control) was thermally treated in a water bath at 75°C. Another part was heat treated in a pressure-pot at 150°C, at a pressure of 0.1 MPa. The next part of samples was subjected to the same temperature, but the pressure was 0.2 MPa. The obtained results indicate that the values of the studied indices were largely affected by thermal processing parameters rather than the animals’ growth rate. The highest contents of total protein and water-soluble collagen were obtained in the case of a temperature of 150°C and the highest pressure (0.2 MPa). Water vapor with increased temperature and pressure also created favorable conditions for obtaining better meat tenderness and more favorable values of the water holding capacity. The latter characteristic appeared to be strongly connected with an increasing amount of water-soluble collagen, which was confirmed by relatively high values of the correlation coefficient between these characteristics. A strong positive correlation was also shown between thermal drip and the total collagen content in meat.

Understanding the Impact of Oven Temperature and Relative Humidity on the Beef Cooking Process

The objective of this study was to evaluate the roles that cooking rate and relative humidity has on the sensory development of beef strip steaks. Thirty USDA Choice beef strip loins were collected from a commercial packing facility. Each strip loin was cut into steaks and randomly assigned to 1 of 6 cooking methods utilizing 2 oven temperatures (80°C and 204°C) and 3 levels of relative humidity [zero (ZH), mid (MH), and high (HH)]. Cooked steaks were used to evaluate internal and external color, Warner-Bratzler and slice shear force, total collagen content, protein denaturation, and trained sensory ratings. Relative humidity greatly reduced cooking rate, especially at 80°C. Steaks cooked at 80°C-ZH had the greatest (P &lt; 0.01) cook loss of all treatments, and cook loss was not affected (P &gt; 0.05). Steaks cooked at 80°C-ZH appeared the most (P &lt; 0.01) well-done and had the darkest (P &gt; 0.01) surface color. Total collagen was greatest (P &lt; 0.01) in steaks cooked with ZH, regardless of oven temperature. Myosin denaturation was not affected (P &gt; 0.05) by treatment. Increased (P = 0.02) sarcoplasmic protein denaturation was observed with ZH and MH, while increased (P = 0.02) actin denaturation was observed only with ZH. Oven temperature did not influence (P &gt; 0.05) protein denaturation. Trained panelists rated steaks the most tender (P &lt; 0.01) when cooked at 80°C and with ZH and MH. Humidity did not affect (P &gt; 0.05) juiciness at 204°C; however, MH and HH produced a juicier (P &lt; 0.01) steak when cooked at 80°C. Humidity hindered (P &lt; 0.01) the development of beefy/brothy and brown/grilled flavors but increased (P = 0.01) metallic/bloody intensity. Lower oven temperatures and moderate levels of humidity could be utilized to maximize tenderness, while minimally affecting flavor development.

Effects of Marbling Texture on Muscle Fiber and Collagen Characteristics

The objective of this study was to evaluate the effects of marbling texture on muscle fiber morphometrics, adipocyte cross-sectional area, perimysial thickness, collagen solubility, and thermal transition temperature of perimysial collagen of beef strip loin steaks from 3 marbling scores. Beef strip loins (n = 117) were selected from three marbling scores [Top Choice (Modest00 – Moderate100 marbling), Low Choice (Small marbling), and Select (Slight marbling)] to equally represent 3 different marbling texture groups: fine, medium, and coarse, via visual appraisal. There were no marbling texture × marbling score interactions for all traits evaluated. Marbling texture impacted (P &lt; 0.05) adipocyte cross-sectional area, where coarse steaks displayed larger adipocytes in comparison to fine marbled steaks, but medium marbled steaks were similar (P &gt; 0.05) to both coarse and fine marbled steaks for the same trait. However, marbling texture did not impact (P &gt; 0.05) perimysial thickness. Marbling texture did not affect collagen traits, as no differences (P &gt; 0.05) were found among marbling texture treatments for soluble collagen, insoluble collagen, and total collagen concentrations. Furthermore, all marbling texture groups (fine, medium, and coarse) were similar (P &gt; 0.05) for the peak thermal transition phase of the perimysial fraction of collagen. Marbling score affected adipocyte size, as Top Choice and Low Choice possessed larger (P &lt; 0.05) adipocytes than Select steaks. There were no differences (P &gt; 0.05) among marbling scores for fiber type; nor were differences found among marbling textures or marbling scores for fiber cross-sectional area. These results indicate that marbling texture does not contribute to differences in collagen characteristics or fiber cross-sectional area that may impact eating quality of beef strip loin steaks.

Effect of ractopamine on physicochemical characteristics and collagen solubility in finishing pigs

The objective of this study was to investigate physicochemical characteristics and collagen solubility of pigs fed with dietary ractopamine (RAC) during finishing period. Thirty of three-way crossbred pigs (Large White × Landrace × Duroc) with an initial bodyweight (BW) of 18.0 ± 0.5 kg were randomly assigned to one to three dietary RAC inclusions (0, 20, or 40 mg/kg) fed during finishing period (60-100 kg BW). Each group consisted of five gilts and five barrows. After 109 day of raising period, all pigs were slaughtered at the average weight of 100 kg. Longissimus dorsi muscle (LD) was taken from each carcass after overnight chill and analyzed for physical characteristics, chemical composition, and collagen solubility. The LD from pigs fed 40 mg/kg of RAC had lower a* value (p &lt; 0.01) than others. Pigs fed with 40 mg/kg of RAC had higher Warner-Bratzler shear force (p &lt; 0.05) than pigs fed without RAC. Pigs fed with dietary RAC had lower glycogen content than control (p &lt; 0.01). While other chemical compositions and collagen solubility of LD were not affected by dietary RAC (p &gt; 0.05). Gilts had larger muscle fibers (p &lt; 0.01), lower glycogen content (p &lt; 0.05), higher content of insoluble collagen (p &lt; 0.05), total collagen (p = 0.054), and collagen solubility (p = 0.054) than barrows.

Rapamycin Inhibits the Growth and Collagen Production of Fibroblasts Derived from Human Urethral Scar Tissue.

Rapamycin can inhibit fibroblast proliferation, collagen accumulation, and urethral stricture in rabbits. Transforming growth factor-beta-1 (TGF-β1) signaling, with downstream recruitment of Smad2, is known to promote fibrosis. This in vitro study examined the effects of rapamycin on fibroblasts derived from human urethral scar tissue (FHUS) and investigated the possible mechanism with respect to regulation of TGF-β1 signaling. FHUS were cultured from urethral scar tissues collected from four patients with urethral stricture. The cells were exposed to different concentrations of rapamycin (0, 10, 20, 40, 80, or 160 ng/ml) for 24 or 48 hours. Cell growth was assessed by the MTT assay. Collagen content was measured based on hydroxyproline levels. The mRNA expressions of Smad2, eIF-4E, and alpha-1 chains of collagen types I and III (Col1α1 and Col3α1) were determined by semiquantitative reverse-transcription PCR. The protein expressions of Smad2, phospho-Smad2, and eIF-4E were evaluated by western blot. Rapamycin caused a concentration-dependent inhibition of FHUS growth at 24 and 48 hours (P < 0.01). Rapamycin decreased total collagen content (P < 0.01), collagen content per 105 cells (P < 0.05), and mRNA expressions of Col1α1 and Col3α1 (P < 0.05) in a concentration-dependent manner. Rapamycin elicited concentration-dependent reductions in the mRNA (P < 0.05) and protein (P < 0.01) expressions of Smad2 and eIF-4E. The two highest concentrations of rapamycin also enhanced phospho-Smad2 levels (P < 0.01). In conclusion, the present study confirmed that rapamycin may reduce the growth and collagen production of FHUS, possibly through inhibition of TGF-β1 signaling.

Gene Network Analysis of Interstitial Macrophages After Treatment with Induced Pluripotent Stem Cells Secretome (iPSC-cm) in the Bleomycin Injured Rat Lung

Idiopathic pulmonary fibrosis (IPF) is a complex disease involving various cell types. Macrophages are essential in maintenance of physiological homeostasis, wound repair and fibrosis in the lung. Macrophages play a crucial role in repair and remodeling by altering their phenotype and secretory pattern in response to injury. The secretome of induced pluripotent stem cells (iPSC-cm) attenuates injury and fibrosis in bleomycin injured rat lungs. In the current study, we evaluate the effect of iPSC-cm on gene expression and phenotype of interstitial macrophage in bleomycin injured rat lungs in vivo. iPSC-cm was intratracheally instilled 7 days after bleomycin induced lung injury and assessed 7 days later and single cell isolation was performed. Macrophages were FACS sorted and microarray analysis was performed. We characterized changes in the rat lung interstitial macrophages using transcriptional profiling. iPSC-cm reduced the total collagen content of the lung and reduced different macrophage populations. Gene set enrichment analysis revealed involvement of three essential pathways (a) immune modulation, (b) branching morphogenesis and (c) canonical Wnt signaling. This study demonstrates that iPSC-cm reduces fibrosis in bleomycin injured rat lung by partially altering the macrophages and regulating their gene expression.

Microvesicles derived from human Wharton's Jelly mesenchymal stem cells ameliorate ischemia-reperfusion-induced renal fibrosis by releasing from G2/M cell cycle arrest.

Studies have demonstrated that microvesicles (MVs) derived from human Wharton's Jelly mesenchymal stromal cells (hWJMSCs) could ameliorate renal ischemia/reperfusion injury (IRI); however, the underlying mechanisms were not clear yet. Here, MVs were isolated and injected intravenously into rats immediately after ischemia of the left kidney, and Erk1/2 activator hepatocyte growth factor (HGF) or inhibitor U0126 was administrated. Tubular cell proliferation and apoptosis were identified by Ki67 or terminal-deoxynucleotidyl transferase-mediated nick end labeling immunostaining. Masson's tri-chrome straining and alpha-smooth muscle actin staining were used for assessing renal fibrosis. The mRNA or protein expression in the kidney was measured by quantitative reverse transcription-PCR or Western blot, respectively. The total collagen concentration was also determined. In vitro, NRK-52E cells that treated with MVs under hypoxia injury and with HGF or U0126 administration were used, and cell cycle analysis was performed. The effects of hWJMSC-MVs on enhancing the proliferation and mitigating the apoptosis of renal cells, abrogating IRI-induced fibrosis, improving renal function, decreasing collagen deposition, and altering the expression levels of epithelial-mesenchymal transition and cell cycle-related proteins in IRI rats were found. In vitro experiment showed that hWJMSC-MVs could induce G2/M cell cycle arrest and decrease the expression of collagen deposition-related proteins in NRK-52E cells after 24 or 48 h. However, U0126 treatment reversed these effects. In conclusion, MVs derived from hWJMSCs ameliorate IR-induced renal fibrosis by inducing G2/M cell cycle arrest via Erk1/2 signaling.

Experimental study of inhibition of tendon fibroblast growth by chitosan through the pathway of TGF-β1/Smad3 mediated by miR-29b

Objective To study the molecular mechanism of chitosan inhibiting fibroblast growth after tendon repair. Methods The rats with Achilles tendon injury were treated with chitosan during operation, and the sliding distance and total collagen content were examined 8 weeks postoperatively. Simultaneously, the fibroblasts in the repaired tendon tissue were separated and treated with different concentrations of chitosan. The survival rate, apoptosis and cell cycle of the cells were examined. In addition, the expression of miRNAs was quantified by real-time PCR and expression of TGF-β1, Smad3 and P21were quantified by western blot. Results Chitosan can increase the tendon gliding distance, reduce the content of collagen fibers, inhibit the growth of fibroblasts and arrest cells in G1 phase of the growth cycle. Chitosan also up-regulated the expression of miR-29b and P21 but down-regulated the levels of TGF-β1and Smad3 in repaired tendon tissue and in vitro fibroblasts. In fibroblasts, miR-29b inhibitor can reverse the expression changes of miR-29b、TGF-β1、Smad3 and P21 induced by chitosan. Conclusion Chitosan can promote the repair of Achilles tendon injury in rats. miR-29b causes fibroblasts staying at G1 phase by down-regulating the expression of TGF-β1and Smad3 and up-regulating the expression of P21. It may be one of the mechanisms of chitosan playing a positive role in the process of tendon repair. Key words: Tendon injuries; Chitosan; Fibroblasts; miR-29b