The Effect of Carbon Dioxide, Lidoflazine, and Deferoxamine Upon Long Term Survival Following Cardiorespiratory Arrest in Rats

This study examined the effect of carbon dioxide, lidoflazine, and deferoxamine therapy upon the 10-day survival incidence and subsequent neurologic function of rats subjected to 7 min of cardiorespiratory arrest with resuscitation. Cardiac arrest (asystole) was induced at time zero by injection of cold, 1% KCl into the left ventricle of ketamine-anesthetized rats pretreated with succinylcholine. Positive pressure ventilation was discontinued at time zero. Cardiopulmonary resuscitation (CPR) was begun at 7 min, and animals with return of spontaneous circulation were entered into the study. Twenty treated rats were ventilated for 1 h with 7% carbon dioxide-93% oxygen and given lidoflazine (2.0 mg/kg, i.v.) and deferoxamine (50 mg/kg, i.v.) 5 min after CPR. Twenty control rats were ventilated for 1 h with 100% oxygen and given lidoflazine vehicle and deferoxamine vehicle. Lidoflazine treatment (1.0 mg/ kg) for the treated group, or lidoflazine vehicle for the control group, was repeated at 8 h postresuscitation. At 2 days postresuscitation, 75% of treated rats vs. 25% of control rats were alive (Chi-square = 10.0, d.f. = 1, P \u3c 0.01), and at 10 days, 60% of treated rats vs. 25% of control rats were alive (Chi-square = 5.01, d.f. = 1, P \u3c 0.05). There was no detectable neurologic deficit among survivors in either group at 15 days. The combination of carbon dioxide, lidoflazine, and deferoxamine, administered after return of spontaneous circulation, is a simple and easily administered treatment regimen that improves the survival incidence without neurologic deficits in this animal model of cardiorespiratory arrest and CPR

Histochemical demonstration of endothelial superoxide and hydrogen peroxide generation in ischemic and reoxygenated rat tissues

Objective: The aims were to test and evaluate two novel and independent histochemical methods for detecting the initial postischemic burst of superoxide and hydrogen peroxide in buffer perfused rat tissues during reflow after 60 min warm ischemia. Methods: The first is a high manganese/diaminobenzidine technique, in which superoxide oxidises Mn2+ to Mn3+, which in turn oxidizes diaminobenzidine to form amber colored polymers, observable by light microscopy. The second is a high iron/diaminobenzidine technique, in which hydrogen peroxide oxidizes diethylenetriaminepenta-acetate chelated Fe2+ to form intermediate species, which in turn oxidize diaminobenzidine similarly to Mn3+. Various isolated organs of the rat were rendered ischemic for 60 min, and reperfused with oxygen or air equilibrated buffers containing diaminobenzidine and either Mn2+ or Fe2+. Tissues were fixed by perfusion with Trump’s solution and processed for light microscopy. Results: Both manganese and iron methods consistently showed the appearance of reaction product on the luminal surfaces of arterial, capillary, and venular endothelial cells in lung, heart, and intestine of the rat during the first 2 to 3 min of reoxygenation after ischemia. The histochemical reactions were nearly absent in non-manganese treated and non-iron-treated controls. Superoxide dismutase strongly inhibited Mn2+/diaminobenzidine reaction product formation and catalase strongly inhibited Fe2+/diaminobenzidine reaction product formation, when tested in specially perfused lung preparations in which these specific antioxidant enzymes were concentrated. Conclusions: These histochemical techniques provide direct, visual evidence that a burst of reactive oxygen species is generated in postischemic rat tissues. The Mn2+/diaminobenzidine and Fe2+/diaminobenzidine techniques permit investigation of the endothelium derived reactive oxygen by simple laboratory procedures available to almost any investigator at low marginal cost. The endothelial oxidants so revealed may be of pathophysiological significance in a variety of cardiovascular disorders

Protection from reperfusion injury in the isolated rat heart by postischaemic deferoxamine and osypurinol administration

The Langendorff isolated rat heart preparation was used to determine the effect of oxypurinol, a xanthine oxidase inhibitor, and deferoxamine, an iron binding agent, on the extent of myocardial reperfusion injury after 60 minutes of ischaemia. Thirty rats were divided into three groups of 10. and an isolated heart preparation made from each rat. The isolated hearts were perfused for 15 minutes with a modified Krebs-Henseleit perfusate solution to permit stabilisation of the preparation. Each heart was then subjected to 60 minutes of total ischaemia at 37°C followed by 60 minutes of reperfusion with either saline treated perfusate, oxypurinol treated perfusate (1.3 mmol/litre), or deferoxamine treated perfusate (0.61 mmol/litre). Reperfusion injury was assessed by the total amount of creatine phosphokinase released into the perfusate, by changes in myocardial vascular resistance, and by morphological examination. The saline treated group released significantly more creatine phosphokinase into the perfusate than either the oxypurinol treated group (p\u3c0.05) or the deferoxamine treated group (p \u3c 0.05). The mean vascular resistance increased for all groups during the 60 minutes of reperfusion compared with that just before ischaemia but was significantly greater in the saline treated group than in the drug treated groups (p \u3c 0.01). Ultrastructural examination of a randomly selected heart from each group after 60 minutes of reperfusion showed pronounced attenuation of mitochondria1 and endoplasmic reticulum swelling, increased maintenance of membrane integrity, and diminished separation of myofilaments in the oxypurinol treated and deferoxamine treated hearts. The mean cross sectional area of mitochondria after 60 minutes of reperfusion was significantly greater in the saline treated group than in the drug treated groups. Thus both oxypurinol and deferoxamine, given after 60 minutes of ischaemia at the onset of reperfusion, can protect the isolated rat heart from reperfusion injury

Peptide hydrogels — a tissue engineering strategy for the prevention of oesophageal strictures

Endoscopic treatment of Barrett’s oesophagus often leads to further damage of healthy tissue causing fibrotic tissue formation termed as strictures. This study shows that synthetic, self-assembling peptide hydrogels (PeptiGelDesign) support the activity and function of primary oesophageal cells, leading to epithelialisation and stratification during in vitro 3D co-culture. Following buffering in culture media, oesophageal stromal fibroblasts (rOSFs) were incorporated into a library of peptide hydrogels, whereas oesophageal epithelial cells (mOECs) were seeded on the surface. Optimal hydrogels (PGD-AlphaProC and PGD-CGD2) supported mOEC viability (>95 %), typical cell morphology (cobblestone-like), a migration rate of 17.4 μm/hr and a migration distance of 364 μm, at 48 hours. Positive expression of typical epithelial markers (ZO-1 and cytokeratins) was witnessed detected using immunocytochemistry at day 3 in culture. Furthermore, optimal hydrogels were identified which supported rOSF viability (> 95%) with homogenous distribution when incorporated into the hydrogels and also promoted the secretion of collagen type I detected using ELISA, at day 7. 3D co-culture model using optimal hydrogels for both cell types supported a stratified epithelial layer (expressing involucrin and AE1/AE3 markers). Findings from this study could lead to the use of peptide hydrogels as a minimally invasive endoscopic therapy to manage oesophageal strictures

Regenerative medicine: today's discoveries informing the future of medical practice

NPJ Regenerative Medicine 2016; 1:16007

Oncological safety of stromal vascular fraction enriched fat grafting in two-stage breast reconstruction after nipple sparing mastectomy: long-term results of a prospective study

OBJECTIVE: Autologous fat transfer (AFT) is commonly used to treat implant palpability and prevent fibrosis and thinning in mastectomy skin flaps. A major limit to this procedure is volume retention over time, leading to the introduction of fat enrichment with stromal vascular fraction (SVF+AFT). Oncological concerns have been raised over the injection of an increased concentration of progenitors cells (ASCs) in the SVF. The aim of the study is to evaluate the long-term cancer recurrence risk of SVF+AFT cases compared to AFT, in patients undergoing Nipple Sparing Mastectomy (NSM). PATIENTS AND METHODS: A prospective study was designed to compare three groups of patients undergoing NSM followed by SVF+AFT, AFT or none (control group), after a two-stage breast reconstruction. Patients were strictly followed-up for at least 5-years from the second stage reconstructive procedure. Loco-regional and systemic recurrence rate were evaluated over time as the primary outcome. Logistic regression was used to investigate which factors were associated with recurrence events and independent variables of interest were: surgical technique, age above 50 years old, lympho-vascular invasion, oncological stage, adjuvant or neoadjuvant chemotherapy, adjuvant radiotherapy and adjuvant hormone therapy. RESULTS: 41 women were included in G1 (SVF+AFT), 64 in G2 (AFT), and 64 in G3 (control group). Loco-regional recurrence rate was 2.4% for G1, 4.7% for G2, and 1.6% for G3. Systemic recurrence was 7.3%, 3.1%, and 3.1%, respectively. Among the variables included, there were no significant risk factors influencing a recurrence event, either loco-regional or systemic. In particular, SVF+AFT (G1) did not increase the oncological recurrence. CONCLUSIONS: Our data suggest that both centrifuged and SVF-enhanced fat transfer have a similar safety level in comparison to patients who did not undergo fat grafting in breast reconstruction after NSM

Regenerative medicine: are we there yet?

NPJ Regen Med 2017 Jan 5; 2:2

Electromechanical characterization of a tissue-engineered myocardial patch derived from extracellular matrix

ObjectiveExtracellular matrix scaffolds have been successfully used for myocardial wall repair. However, regional functional evaluation (ie, contractility, electrical conductivity) of the extracellular matrix scaffold during the course of remodeling has been limited. In the present study, we evaluated the remodeled scaffold for evidence of electrical activation.MethodsThe extracellular matrix patch was implanted into the porcine right ventricular wall (n = 5) to repair an experimentally produced defect. Electromechanical mapping was performed with the NOGA system (Biosense Webster Inc, Diamond Bar, Calif) 60 days after implantation. Linear local shortening was recorded to assess regional contractility. After sacrifice, detailed histologic examinations were performed.ResultsHistologic examinations showed repopulation of the scaffold with cells, including a monolayer of factor VIII–positive cells in the endocardial surface and multilayered α-smooth muscle actin–positive cells beneath the monolayer cells. The α-smooth muscle actin–positive cells tended to be present at the endocardial aspect of the remodeled scaffold and at the border between the remodeled scaffold and the normal myocardium. Electromechanical mapping demonstrated that the patch had low-level electrical activity (0.56 ± 0.37 mV; P < .0001) in most areas and moderate activity (2.20 ± 0.70 mV; P < .0001) in the margin between the patch and the normal myocardium (7.58 ± 2.23 mV).ConclusionsThe extracellular matrix scaffolds were repopulated by α-smooth muscle actin–positive cells 60 days after implantation into the porcine heart. The presence of the cells corresponded to areas of the remodeling scaffold that showed early signs of electrical conductivity

Amine functionalization of cholecyst-derived extracellular matrix with generation 1 PAMAM dendrimer

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Biomacromolecules, copyright © American Chemical Society after peer review. To access the final edited and published work, see http://pubs.acs.org/doi/pdf/10.1021/bm701055k.A method to functionalize cholecyst-derived extracellular matrix (CEM) with free amine groups was established in an attempt to improve its potential for tethering of bioactive molecules. CEM was incorporated with Generation-1 polyamidoamine (G1 PAMAM) dendrimer by using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysuccinimide cross-linking system. The nature of incorporation of PAMAM dendrimer was evaluated using shrink temperature measurements, Fourier transform infrared (FTIR) assessment, ninhydrin assay, and swellability. The effects of PAMAM incorporation on mechanical and degradation properties of CEM were evaluated using a uniaxial mechanical test and collagenase degradation assay, respectively. Ninhydrin assay and FTIR assessment confirmed the presence of increasing free amine groups with increasing quantity of PAMAM in dendrimer-incorporated CEM (DENCEM) scaffolds. The amount of dendrimer used was found to be critical in controlling scaffold degradation, shrink temperature, and free amine content. Cell culture studies showed that fibroblasts seeded on DENCEM maintained their metabolic activity and ability to proliferate in vitro. In addition, fluorescence cell staining and scanning electron microscopy analysis of cell-seeded DENCEM showed preservation of normal fibroblast morphology and phenotype

Extracellular Matrix Membrane Induces Cementoblastic/Osteogenic Properties of Human Periodontal Ligament Stem Cells

Objective: Periodontitis affects nearly 90% of adults over the age of 70, resulting to periodontal tissue infection, destruction, and ultimately tooth loss. Guided tissue regeneration (GTR) is a method widely used to treat severe periodontal disease, and involves placement of an occlusive barrier to facilitate regeneration of the damaged area by periodontal ligament stem cells (PDLSCs). In this study, we evaluate natural extracellular matrix (ECM) as a scaffold material to provide a suitable microenvironment to support the proliferation, differentiation, and tissue-regenerating properties of PDLSCs.Design: The viability, proliferation, apoptosis, and migration of PDLSCs cultured on ECM membrane, that was isolated from porcine urinary bladders, were compared with those cultured on type I collagen membrane, a commonly used scaffold in GTR. To evaluate the effects of ECM vs. type I collagen on the tissue-regenerating properties of PDLSCs, the bio-attachment and cementoblastic/osteogenic differentiation of PDLSCs were evaluated.Results: Incubation of PDLSCs with ECM resulted in increased viability, proliferation, and reduced apoptosis, compared with type I collagen treated PDLSCs. Co-culture with ECM membrane also increased the migration and bio-attachment of PDLSCs. Incubation of PDLSCs with ECM membrane increased expression of the cementoblastic/osteogenic differentiation markers BSP, RUNX2, ALP, OPN, OCN, and periostin.Conclusion: ECM membrane enhances the proliferation and regenerative properties of PDLSCs, indicating that ECM membrane can serve as a suitable scaffold in the application of GTR to treat periodontal disease