Perfusion Method Research Articles

Activation of GLP-1 receptor signalling alleviates cellular stresses and improves beta cell function in a mouse model of Wolfram syndrome.

Loss of functional beta cells results in a gradual progression of insulin insufficiency in Wolfram syndrome caused by recessive WFS1 mutations. However, beta cell dysfunction in Wolfram syndrome has yet to be fully characterised, and there are also no specific treatment recommendations. In this study, we aimed to characterise beta cell secretory defects and to examine the potential effects of a glucagon-like peptide-1 (GLP-1) receptor agonist on diabetes in Wolfram syndrome. Insulin secretory function was assessed by the pancreatic perfusion method in mice used as a model of Wolfram syndrome. In addition, granule dynamics in living beta cells were examined using total internal reflection fluorescence microscopy. Acute and chronic effects of exendin-4 (Ex-4) on glucose tolerance and insulin secretion were examined in young Wfs1-/- mice without hyperglycaemia. Molecular events associated with Ex-4 treatment were investigated using pancreatic sections and isolated islets. In addition, we retrospectively observed a woman with Wolfram syndrome who had been treated with liraglutide for 24weeks. Treatment with liraglutide ameliorated our patient's glycaemic control and resulted in a 20% reduction of daily insulin dose along with an off-drug elevation of fasting C-peptide immunoreactivity. Glucose-stimulated first-phase insulin secretion and potassium-stimulated insulin secretion decreased by 53% and 59%, respectively, in perfused pancreases of 10-week-old Wfs1-/- mice compared with wild-type (WT) mice. The number of insulin granule fusion events in the first phase decreased by 41% in Wfs1-/- beta cells compared with WT beta cells. Perfusion with Ex-4 increased insulin release in the first and second phases by 3.9-fold and 5.6-fold, respectively, in Wfs1-/- mice compared with perfusion with saline as a control. The physiological relevance of the effects of Ex-4 was shown by the fact that a single administration potentiated glucose-stimulated insulin secretion and improved glucose tolerance in Wfs1-/- mice. Four weeks of administration of Ex-4 resulted in an off-drug amelioration of glucose excursions after glucose loading in Wfs1-/- mice, with insulin secretory dynamics that were indistinguishable from those in WT mice, despite the fact that there was no alteration in beta cell mass. In association with the functional improvements, Ex-4 treatment reversed the increases in phosphorylated eukaryotic initiation factor (EIF2α) and thioredoxin interacting protein (TXNIP), and the decrease in phosphorylated AMP-activated kinase (AMPK), in the beta cells of the Wfs1-/- mice. Furthermore, Ex-4 treatment modulated the transcription of oxidative and endoplasmic reticulum stress-related markers in isolated islets, implying that it was able to mitigate the cellular stresses resulting from Wfs1 deficiency. Our study provides deeper insights into the pathophysiology of beta cell dysfunction caused by WFS1 deficiency and implies that activation of the GLP-1 receptor signal may alleviate insulin insufficiency and aid glycaemic control in Wolfram syndrome.

The experimental study of endothelial progenitor cells in rat liver decellularized scaffold

Objective To engineer intact whole rat liver decellularized scaffolds and repopulated bone marrow-derived endothelial progenitor cells (EPCs) into the scaffolds by continous perfusion technology thus providing experimental support for the vasculation of decellularized liver scaffolds in liver engineering. Methods Decellularized liver scaffolds were obtained by perfusing method. The composition and structure was examed by hematoxylin-eosin staining (HE) and immunohistochemistry. Bone marrow derived EPCs were characterised by immunofluorescence of CD31, CD133 and Vascular endothelial growth factor (VEGF). EPCs were recellularized into the decellularized scaffolds by portal-vein infusion method and cultured by the dynamic circulation perfusion device. After cultivation, HE staining and immunofluorescence of CD31 were conducted to observe the growth situation of EPCs in the scaffolds. Results The rat decellulariezd liver scaffolds were successfully obtained by perfusion method. Histological staining demonstrated the remove of cellular component and the reservation of extracellular cell matrix. Immunohistochemistry staining demonstrated the retention of collagen I and laminin. The primary cultured EPCs were postive for CD31, CD133 and VEGF. The circulation perfusion device was composed of a peristaltic pump, oxygenator, chamber and the convey tubes. HE staining and immunofluorence revealed that EPCs can be located around the blood vessel wall. Conclusion The primary cultured EPCs can grew around the blood vessel wall of the decellularized liver scaffolds by circulation perfusion method. Key words: Liver; Decellularized scaffolds; Circulation perfusion culture; Vascularization

Basic research on uterus transplantation in nonhuman primates in Japan.

Uterus transplantation (UTx) is now a potential option for women with uterine factor infertility to have a child. However, UTx is still in an experimental stage and basic animal studies including in nonhuman primates are needed for accumulation of data that will provide important information for establishment of UTx in humans. Herein, we summarized our experiences using cynomolgus macaques, with the goal of promoting further development of UTx studies in nonhuman primates. Our basic studies using cynomolgus macaques were summarized, including the results of other teams in nonhuman primates. Our team in Japan launched UTx research in 2009 using cynomolgus macaques and has accumulated a large archive of results in the UTx research field, including examination of uterine blood flow, surgical procedures of autologous and allogeneic UTx, organ perfusion methods in deceased donor models, immunological response and rejection and ischemia/reperfusion injury. We achieved the first delivery after autologous UTx in primates and the first periodic recovery of menstruation after allogeneic UTx in nonhuman primate models. Results from animal studies, including those in nonhuman primates, provide the basis for clinical application of UTx. Therefore, our accumulated data since 2009 and our basic experience in cynomolgus macaque are meaningful for future UTx trials in Japan. In addition, more validation in nonhuman primate models is needed for resolution of medical issues and further development of UTx in humans, despite clinical application of UTx in several countries.

<b>Development of equipment for decellularization using the perfusion method

This study describes the development of equipment capable of transporting decellularization fluid through an organ by perfusion to create an in vitro acellular scaffold that maintains the three-dimensional architecture of the organ. The equipment was designed to be compatible with several existing decellularization protocols and can be used to modify the flow rate, perfusion pressure, and temperature in each decellularization protocol as well as the technique of cannulation of the organ in a controlled manner. The device was tested with chicken hearts and efficiently accomplished decellularization using the perfusion method. Further amendments can be made to improve the efficiency of the decellularization of diverse organs. It is expected that this equipment will aid in the characterization and improvement of the decellularization process and may have future applications in the process of recellularization of different organs.

1. Development of bioengineered xenogeneic decellularized humanized liver for the development of extracorporeal liver for the treatment of acute liver failure

Background and Aims: Acute as well as chronic liver failures are major fatal problems, which lead up to 60–80% mortality every year. In acute condition, systemic inflammation and accumulation of toxic compounds (ammonia) in liver results in multi-organ failure which leads to hepatic encephalopathy. Liver transplantation is limited due to timely unavailability of enough donors, post-transplantation complications and high cost involvement. The present study demonstrates a promising strategy of using bioartificial extracorporeal liver support system prepared through decellularization and repopulation of xenogenic liver with human hepatic progenitor cells (hHPCs). Methods: Xenogenic liver was decellularized by perfusion method using retrograde change of detergents and other chemicals. Removal of nuclear components, retention of ECM and vascular integrity of whole decellularized liver was identified. DiD-labeled hHPCs were infused to identify the cells distribution and engraftment efficiency. Repopulation of hHPCs within the decellularized liver scaffold was determined by SEM analysis whereas the functional activity of repopulated cells was determined by ammonia detoxification experiment by urea quantification. Results: The decellularized liver vascular network was able to withstand fluid flow that entered through a central inlet vessel, branched into an extensive capillary bed, and coalesced into a single outlet vessel. Repopulated hHPCs showed homogeneous distribution within the decellularized liver scaffold showing defined vascular tree with multiple branching and residual niches of proliferating cells. Ammonia detoxification showed the functional activity and future applicability of repopulated humanized liver scaffold as extra-corporeal natural organ support system. Conclusions: This study demonstrate an innovative technology for bioengineering humanized extra- corporeal liver system as better approach for ammonia detoxification as temporary support to the failing liver. The authors have none to declare.

Impact of In Situ Cold Perfusion of Kidney Grafts during Vascular Anastomosis

This study investigated simple in situ perfusion of a kidney to reduce its susceptibility to warm ischemia in rodent and porcine ischemic kidney models. Organ temperature was managed via simple continuous perfusion using newly developed thermal barrier sheet (TBS). ATP retention following cold perfusion with extracellular-trehalose-Kyoto (ETK) solution was investigated in a rat model. Materials and Methods Kidneys from adult male genetically modified luciferase transgenic (Luc Tg) rats were removed. The renal artery/vein underwent simple cold preservation at 4°C for 5 to 18 hours using ETK solution. After cold storage, the kidney was transplanted into a dummy living body that was heated to 37°C and perfused with ETK solution using a drip. The surface temperature (Ts) was measured with an infrared camera and ATP luminescence in organs was observed for 60 minutes. In porcine, the renal artery and vein were clamped within the abdominal cavity and the left kidney was prepared as a donor organ after 25 minutes of exposure to warm ischemia. The kidney was perfused via the renal artery at 4°C using ETK solution and subsequently underwent cold preservation (Fig1.a). We continued the surgery, while the kidney continued to undergo cold perfusion (Fig2.b) using a TBS. Continuous suturing was performed and the cannula was removed immediately after reflux. Blood flow then resumed. Control kidneys were sutured without undergoing perfusion. The time required for anastomosis was 1 hour, during which time the Ts of the kidney was measured. After resuming blood flow, the right kidney was removed. Following transplantation, blood and tissue were collected. Results Using a TBS and cold reflux in the dummy body, the rodent kidney Ts was maintained at 15°C or less for more than 30 minutes. During perfusion with the conventional method, the kidney Ts was at least 28°C immediately following removal. In the case of cold perfusion and TBS, the ATP level was elevated by approximately 4-fold that of pre-removal levels during preservation. Meanwhile, while using the conventional method, the ATP level was 0.9-fold of the pre-removal level during cold preservation. The porcine kidney Ts in the control group increased to 32°C during transplantation. Whereas, in the cold perfusion group, transplantation was performed while the kidney Ts was maintained at 24°C. The primary non-function rate after transplantation was 60% in the control group and 25% in the cold perfusion group. Discussion The cold perfusion method using a TBS can maintain the temperature and ischemic time in all organs obtained after cardiac death. In addition, the preclinical porcine model is a useful renal transplantation graft model that does not exhibit rejection and can be used to identify appropriate constant temperature perfusion solutions. Conclusions By developing this perfusion system, we believe that we can promote research toward practical application of machine perfusion.

The fate of hepatocyte cell line derived from a liver injury model with long-term in vitro passage

Orthotopic Liver Transplantation (OLT) is the therapy of choice for the treatment of end-stage liver disease, but the severe shortage of organ donors, complex and expensive surgical procedure and increased mortality of prospective organ recipient limit the use of OLT. To overcome this problem the technique of hepatocyte transplantation has been considered as an alternative to OLT. Hepatocyte transplantation is less invasive, cost effective cryo-preservable and can be distributed from single donor to multiple recipients. In this study we have established and characterize the hepatocyte cell line possessing the morphological and functional characteristics of hepatocytes from chemically injured liver. Hence we hypothesized that the hepatocyte cell line derived from liver injury can be used for hepatocyte transplantation. To induce the priming of hepatocyte, mice were fed with 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet for 3 weeks and hepatocytes were obtained by two step collagenase perfusion method, hepatocytes hence obtained was expended by >300 passages and tested for various hepatocyte specific functions. The cell line derived from liver injury model retains morphological and functional characteristics of hepatocytes in long-term in vitro culture. These cells transplanted to mice showed significant survival rate. The Hepatocyte cell line derived from liver injury model were used for hepatocyte transplantation and showed the significantly higher survival rate.

Time-shifting effects of methylphenidate on daily rhythms in the diurnal rodent Arvicanthis ansorgei.

People suffering of attention-deficit/hyperactivity disorder (ADHD) and treated with the psychostimulant methylphenidate (MPH) show sleep-wake cycle and daily rhythm alterations despite the beneficial effects of MPH on behavioral symptoms (i.e., hyperactivity, attention). In nocturnal rodents (i.e., mice), chronic exposure to MPH alters the neural activity of the circadian clock in the suprachiasmatic nucleus (SCN), behavioral rhythms, and the sleep-wake cycle. Here, we studied the effects of MPH on daily rhythms of behavior and body temperature of the diurnal rodent Arvicanthis ansorgei. Under a light-dark cycle, chronic exposure to MPH in drinking water delayed the onset of both activity and body temperature rhythms. Interestingly, delays were larger when MPH access was restricted to the first 6h of the light phase (i.e., activity phase) of the 24-h cycle. Since MPH effects are dependent on animal's fluid intake, in a last experiment, we controlled the time and dose of MPH delivery in Arvicanthis using an intraperitoneal perfusion method. Similarly to the experiment with MPH in drinking water, Arvicanthis showed a delay in the onset of general activity and body temperature when MPH infusions, but not vehicle, were during the first 6h of the light phase. This study indicates that MPH alters daily rhythms in a time-dependent manner and proposes the use of a diurnal rodent for the study of the effects of MPH on the circadian clock. Knowing the circadian modulation on the effects of MPH in behavior could give new insights in the treatment of ADHD.

A simple antegrade perfusion method for isolating viable single cardiomyocytes from neonatal to aged mice.

The aim of this study was to establish a simple and reproducible antegrade perfusion method for isolating single viable mouse heart cells and to determine the standard practical protocols that are appropriate for mice of various ages. Antegrade perfusion was performed by injecting perfusate from near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. This could thoroughly perfuse the myocardium through the coronary circulation. All procedures were carried out on a prewarmed heater mat under a microscope, which allows for the processes of injection and perfusion to be monitored. With appropriate adjustment of the size of the injection needle, the composition and amount of enzyme solution and the perfusion flow rate, this antegrade perfusion method could be applied to the hearts of neonatal to aged mice. We examined the morphological characteristics and electrophysiological properties of the isolated ventricular and atrial myocytes and found that these cells were mostly identical to those obtained with the traditional Langendorff‐based retrograde perfusion method. Interstitial nonmyocytes, such as cardiac progenitor cells, were also isolated simultaneously from the supernatant fraction of the centrifugation, similar to the retrograde perfusion method. The results suggest that single heart cells can be well isolated with high degree of quality by the present antegrade perfusion method, regardless of the age of the mouse.

Effects of Subnormothermic Perfusion Before Transplantation for Liver Grafts from Donation After Cardiac Death: A Simplified Dripping Perfusion Method in Pigs

BackgroundLiver transplantation from donors after cardiac death (DCD) provides a solution to the donor shortage. However, DCD liver grafts are associated with a high incidence of primary graft nonfunction. We investigated the effectiveness of subnormothermic porcine liver perfusion, before transplantation from DCD, on graft viability. MethodsLandrace pigs (25–30 kg) were randomly allocated to 3 groups (5 per group): heart-beating (HB) graft, transplanted after a 4-hour period of cold storage (CS); DCD graft, retrieved 20 minutes after apnea-induced cardiac arrest (respiratory withdrawal) and transplanted after a 4-hour period of CS; and subnormothermic ex vivo liver perfusion (SELP) graft, retrieved in the same manner as the DCD graft but perfused with a subnormothermic oxygenated Krebs-Henseleit buffer (21–25°C, 10–15 cm H2O) for 30 minutes in a simplified dripping manner, without a machine perfusion system, after the 4-hour period of CS, and subsequently transplanted. ResultsAlthough all animals in the HB group survived for >7 days, all animals in the DCD group died within 12 hours after transplantation. In the SELP group, 2 recipients survived for >7 days and another 2 recipients were killed on day 5. The survival rate was significantly better for SELP than for DCD grafts (P = .0016). The values of tumor necrosis factor α were not significantly different between the SELP and HB groups. Preserved structure of the parenchyma was observed in the SELP group on histologic examination. ConclusionsA simplified subnormothermic perfusion before liver transplantation is expected to improve graft viability and survival.

Multidrug Resistance Reversal Activity of Some New Dihydropyridines Studied by IN SITU Single-Pass Intestinal Perfusion (SPIP) Method in Rat

P-glycoprotein (P-gp) mediated efflux affects the pharmacokinetics of several drugs. By analogy to verapamil, 1,4-dihydropyridines (DHPs) have been widely studied as P-gp inhibitors. Previously, we have reported on two new DHPs: IA1(A) and IIA5(B) as inhibitors of human MRP1, an efflux protein closely related to P-gp. The aim of the present study was to investigate the inhibitory effects of these two compounds on intestinal P-gp using the method of in situ single-pass intestinal perfusion (SPIP) in rat. According to this, the intestinal absorption of zidovudine (a P-gp substrate) was studied in anaesthetized rat jejunum in the absence and presence of DHPs IA1(A) and IIA5(B) (2 mg/kg). Verapamil (0.8 mg/kg), a well-known P-gp inhibitor, was employed as a standard. Zidovudine solution (200 ig/mL) in phosphate buffer (pH 7.4) was perfused through the jejunal segment, the perfusate concentrations were quantified by HPLC, and the permeability coefficient (Peff) and fraction absorbed (Fabs) were calculated. Phenol red was used as a non-absorbable marker to correct water flux through the segment. In rats pretreated with compounds IA1 and IIA5, Peff and Fabs of zidovudine were found to be 0.1669 ± 0.12 cm/sec, 0.2035 ± 0.18 and 0.2798 ± 0.12 cm/sec, 0.3015 ± 0.14, respectively, and were comparable to those of the standard (Peff = 0.462713 ± 0.3 cm/sec, Fabs = 0.511835 ± 0.14). The differences between IA1, IIA5 and the standard were evaluated using ANOVA and found to be statistically significant (P < 0.05). Compounds IA1 and IIA5 have a modulating effect on intestinal P-gp. Compound IIA5 was relatively more potent P-gp inhibitor and, quite interestingly, the results were in agreement with our earlier in silico and in vitro studies.

Effects of non‐esterified fatty acids on the expression and secretion of angiopoietin‐like protein 4 and fibroblast growth factor 21 in calf hepatocytes cultured in vitro

IntroductionNegative energy balance (NEB) is considered as the pathological basis of energy metabolic disorders, such as fatty liver and ketosis, in periparturient dairy cows. An elevated serum non‐esterified fatty acids (NEFA) concentration is one of the most important indicators of NEB. Angiopoietin‐like protein 4 (ANGPTL4) and fibroblast growth factor 21 (FGF21) have been identified as hepatokines, which involved in regulation of metabolic adaptations. In dairy cows, most circulating ANGPTL4 and FGF21 originate from the liver. However, the direct effects of NEFA on the expression and secretion of ANGPTL4 and FGF21 in bovine hepatocytes are not entirely clear. The purpose of the present study was to investigate the effects of different NEFA concentrations on the expression and secretion of these two hepatokines in calf hepatocytes cultured in vitro.MethodsBovine hepatocytes were cultured using the collagenase perfusion method. After culture for 72 h, the hepatocytes were treated with NEFA at final concentrations of 0 (control), 0.6, 1.2, 1.8 and 2.4 mmol/L. After 24 h of continuous culture, the mRNA and protein expression levels of ANGPTL4 and FGF21 in the hepatocytes were determined by real‐time PCR and Western blot, respectively. ANGPTL4 and FGF21 secretions in the supernatant were determined by enzyme‐linked immunosorbent assay (ELISA).ResultsThe results showed similar levels of ANGPTL4 expression and secretion following treatment with 0.6 and 1.2 mmol/L NEFA, which were higher than those observed for the controls (P&lt;0.05). ANGPTL4 expression and secretion were also similar at 1.8 and 2.4 mmol/L NEFA and significantly higher than those observed for controls (P&lt;0.01). The expression and secretion of FGF21 at 0.6 mmol/L NEFA‐treated hepatocytes was higher than that of the control group (P&lt;0.05). The FGF21 expression and secretion were similar at 1.2, 1.8 and 2.4 mmol/L NEFA‐treated hepatocytes and significantly higher than those observed for controls (P&lt;0.01).ConclusionThe results of this study indicate that high concentrations of NEFA significantly increase the expression and secretion of ANGPTL4 and FGF21 in bovine hepatocytes cultured in vitro in a dose‐dependent manner. Specifically, NEFA promotes the expression and secretion of ANGPTL4 and FGF21, which may worsen fat mobilization under NEB conditions and thereby lead to the pathological processes of energy metabolism disorders in dairy cows in the peripartum period. However, further studies are needed to elucidate the underlying mechanisms.Support or Funding InformationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Preparation of whole-kidney acellular matrix using peristaltic pump perfusion and identification the composition of matrix

Objective To make a whole-kidney acellular matrix scaffold through the method of peristaltic pump perfusion and to identify whether the kidney acellular matrix reserves extracellular matrix proteins or not. Methods The male Sprague-Dawley (SD) rats, 200~250 g. Intravenous cathers were injected into the renal artery to establish the channels for perfusion, followed by the perfusion of decellularization solution by using peristaltic pumps. After the decellularization, the acellular matrix was observed under microscope after hematoxylin and eosin(HE) staining and fluorescence microscope after DAPI staining. Immunohistochemistry was performed to identify the composition of kidney acellualr matrix. Results The result of HE staining and DAPI staining demonstrate the whole removal of cellular material in kidney acelluar matrix. Immunohistochemistry confirmed the preservation of the natural expression type of extracellular matrix proteins including collagen types I and IV, fibronectin and laminin. Conclusions Peristaltic pump perfusion is a successful method to make the kidney acellular matrix, and this scaffold retains the critical proteins of natural extracellular matrix. Therefore the kidney acellular matrix is an ideal natural scaffold for renal tissue engineering. Key words: Kidney Failure; Peristalsis; Extracellular Matrix

Biowaiver or Bioequivalence: Ambiguity in Sildenafil Citrate BCS Classification.

The aim of the present study is to contribute to the scientific characterization of sildenafil citrate according to the Biopharmaceutics Classification System, following the World Health Organization (WHO) guidelines for biowaivers. The solubility and intestinal permeability data of sildenafil citrate were collected from literature; however, the experimental solubility studies are inconclusive and its "high permeability" suggests an API in the borderline of BCS Class I and Class II. The pH-solubility profile was determined using the saturation shake-flask method over the pH range of 1.2-6.8 at a temperature of 37°C in aqueous media. The intestinal permeability was determined in rat by a closed-loop in situ perfusion method (the Doluisio technique). The solubility of sildenafil citrate is pH-dependent and at pH 6.8 the dose/solubility ratio obtained does not meet the WHO criteria for "high solubility." The high permeability values obtained by in situ intestinal perfusion in rat reinforce the published permeability data for sildenafil citrate. The experimental results obtained and the data available in the literature suggest that sildenafil citrate is clearly a Class II of BCS, according to the current biopharmaceutics classification system and WHO guidance.

Interleukin‑10 promotes primary rat hepatic stellate cell senescence by upregulating the expression levels of p53 and p21.

Liver fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) components, and activated hepatic stellate cells (HSCs) are a primary source of ECM. Several studies have revealed that the induction of HSC senescence may reduce liver fibrosis. The effect of interleukin-10 (IL-10) on the senescence of activated HSCs is not fully understood. Therefore, the present study examined its effects and potential mechanisms in activated primary rat HSCs. Collagenase perfusion and density gradient centrifugation methods were used to isolate rat HSCs. HSCs were identified by autofluorescence, Oil Red O staining and immunocytochemical analysis. Activated HSCs were treated with 0, 10, 20 or 40 ng/ml IL-10 for 24 h. Senescence-associated β-galactosidase (SA-β-Gal) staining, flow cytometry analysis and a cell counting kit-8 assay were performed to detect the senescence, apoptosis and viability of rat HSCs, respectively. Reverse transcription-quantitative polymerase chain reaction, western blot analysis and enzyme linked immunosorbent assays were used to detect the expression of senescence-associated proteins and cytokines. Freshly isolated rat HSCs exhibited a striking blue-green autofluorescence and HSC retinoid droplets were stained bright red by Oil Red O. Immunocytochemical analysis demonstrated the cytoplasmic expression of HSC markers desmin and α-smooth muscle actin. The number of SA-β-Gal positive HSCs, the apoptotic rate and the expression levels of p53, p21 and tumor necrosis factor-α were significantly increased following IL-10 treatment. HSC viability and IL-6 and IL-8 expression levels were significantly decreased compared with the control group. In summary, primary rat HSCs were successfully isolated and IL-10 was demonstrated to promote the senescence of activated primary rat HSCs through the upregulation of p53 and p21 expression.

Assessment of static and perfusion methods for decellularization of PCL membrane-supported periodontal ligament cell sheet constructs

ObjectivesDecellularization aims to harness the regenerative properties of native extracellular matrix. The objective of this study was to evaluate different methods of decellularization of periodontal ligament cell sheets whilst maintaining their structural and biological integrity. DesignHuman periodontal ligament cell sheets were placed onto melt electrospun polycaprolactone (PCL) membranes that reinforced the cell sheets during the various decellularization protocols. These cell sheet constructs (CSCs) were decellularized under static/perfusion conditions using a) 20 mM ammonium hydroxide (NH4OH)/Triton X-100, 0.5% v/v; and b) sodium dodecyl sulfate (SDS, 0.2% v/v), both +/− DNase besides Freeze–thaw (F/T) cycling method. CSCs were assessed using a collagen quantification assay, immunostaining and scanning electron microscopy. Residual fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were assessed with Bio-plex assays. ResultsDNA removal without DNase was higher under static conditions. However, after DNase treatment, there were no differences between the different decellularization methods with virtually 100% DNA removal. DNA elimination in F/T was less efficient even after DNase treatment. Collagen content was preserved with all techniques, except with SDS treatment. Structural integrity was preserved after NH4OH/Triton X-100 and F/T treatment, while SDS altered the extracellular matrix structure. Growth factor amounts were reduced after decellularization with all methods, with the greatest reduction (to virtually undetectable amounts) following SDS treatment, while NH4OH/Triton X-100 and DNase treatment resulted in approximately 10% retention. ConclusionsThis study showed that treatment with NH4OH/Triton X-100 and DNase solution was the most efficient method for DNA removal and the preservation of extracellular matrix integrity and growth factors retention.

Paeonol Nanoemulsion for Enhanced Oral Bioavailability: Optimization and Mechanism

The aim of this work was to optimize a nanoemulsion formulation and explain its absorption mechanism in improving the oral bioavailability of paeonol. The bioavailability of paeonol was compared between paeonol nanoemulsion group and paeonol suspension group. The in situ single-pass intestine perfusion method, in vitro everted gut sacs method, Western blot analysis and Caco-2 cell transport studies were used to investigate the absorption mechanism of nanoemulsion. Nanoemulsion was proved to enhance the bioavailability of paeonol. P-glycoprotein (P-gp)mediated efflux might be the main reason affecting the oral absorption of paeonol. The prepared nanoemulsion prevented the P-gp-mediated efflux and enhanced the bioavailability of paeonol. The overall results revealed that nanoemulsion was an effective vehicle to improve the oral bioavailability of paeonol which resulted from the prevention of P-gp efflux probably.

Arterial spin labeling: a technical overview.

Arterial spin labeling (ASL) is a non-invasive magnetic resonance imaging perfusion method based on changes in net-magnetization of blood water. The absence of contrast use and ionizing radiation, renders ASL valuable in hyper-acute settings as a monitoring tool for repeated dynamical measurements during and after intervention, and for patients with known co-morbidities. This text provides a short methodological introduction to ASL and contrasts it with traditional contrast-enhanced perfusion imaging. The review focused on sequence usefulness in the clinical setting of acute cerebral ischemia investigation.

Decellularization of placentas: establishing a protocol.

Biological biomaterials for tissue engineering purposes can be produced through tissue and/or organ decellularization. The remaining extracellular matrix (ECM) must be acellular and preserve its proteins and physical features. Placentas are organs of great interest because they are discarded after birth and present large amounts of ECM. Protocols for decellularization are tissue-specific and have not been established for canine placentas yet. This study aimed at analyzing a favorable method for decellularization of maternal and fetal portions of canine placentas. Canine placentas were subjected to ten preliminary tests to analyze the efficacy of parameters such as the type of detergents, freezing temperatures and perfusion. Two protocols were chosen for further analyses using histology, scanning electron microscopy, immunofluorescence and DNA quantification. Sodium dodecyl sulfate (SDS) was the most effective detergent for cell removal. Freezing placentas before decellularization required longer periods of incubation in different detergents. Both perfusion and immersion methods were capable of removing cells. Placentas decellularized using Protocol I (1% SDS, 5 mM EDTA, 50 mM TRIS, and 0.5% antibiotic) preserved the ECM structure better, but Protocol I was less efficient to remove cells and DNA content from the ECM than Protocol II (1% SDS, 5 mM EDTA, 0.05% trypsin, and 0.5% antibiotic).

Langendorff Perfusion Method as an Ex Vivo Model to Evaluate Heart Function in Rats.

The Langendorff Perfused Heart Model is an experimental procedure developed at the end of the nineteenth century by Oskar Langendorff. In this procedure, an excised heart has a cannula inserted into its aorta so that the heart can be retrogradely perfused via the coronary artery. The procedure has been improved in recent times, and these improvements are used to evaluate the direct effect of medication on the heart as well as the effect of ischemia-reperfusion injury on heart function. In this chapter, we describe protocols for evaluating heart function in Langendorff perfused rat heart.