at SciVerse ScienceDirect
Biomaterials 34 (2013) 30e41
Contents lists available
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
The blood and vascular cell compatibility of heparin-modified ePTFE vascular
grafts
Ryan A. Hoshi a, Robert Van Lith a, Michele C. Jen a, Josephine B. Allen b, Karen A. Lapidos a,
Guillermo Ameer a,c,*
a Biomedical Engineering Department, Northwestern University, Evanston, IL 60208, USA
b Material Science and Engineering Department, University of Florida, Gainesville, FL 32611, USA
c Department of Surgery, Feinberg School of Medicine, Chicago, IL 60611, USA
a r t i c l e i n f o
Article history:
Received 16 July 2012
Accepted 21 September 2012
Available online 12 October 2012
Keywords:
Vascular graft
Elastomer
Endothelial cell
Progenitor cell
Smooth muscle cell
Heparin
Hemocompatibility
Aminated poly(1,8-octanediol-co-citrate)
(POC)
* Corresponding author.
E-mail address: [email protected] (G. A
0142-9612/$ e see front matter � 2012 Elsevier Ltd.
http://dx.doi.org/10.1016/j.biomaterials.2012.09.046
a b s t r a c t
Prosthetic vascular grafts do not mimic the antithrombogenic properties of native blood vessels and
therefore have higher rates of complications that involve thrombosis and restenosis. We developed an
approach for grafting bioactive heparin, a potent anticoagulant glycosaminoglycan, to the lumen of ePTFE
vascular grafts to improve their interactions with blood and vascular cells. Heparin was bound to ami-
nated poly(1,8-octanediol-co-citrate) (POC) via its carboxyl functional groups onto POC-modified ePTFE
grafts. The bioactivity and stability of the POC-immobilized heparin (POCeHeparin) were characterized
via platelet adhesion and clotting assays. The effects of POCeHeparin on the adhesion, viability and
phenotype of primary endothelial cells (EC), blood outgrowth endothelial cells (BOECs) obtained from
endothelial progenitor cells (EPCs) isolated from human peripheral blood, and smooth muscle cells were
also investigated. POCeHeparin grafts maintained bioactivity under physiologically relevant conditions
in vitro for at least one month. Specifically, POCeHeparin-coated ePTFE grafts significantly reduced
platelet adhesion and inhibited whole blood clotting kinetics. POCeHeparin supported EC and BOEC
adhesion, viability, proliferation, NO production, and expression of endothelial cell-specific markers von
Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin). Smooth muscle cells cultured
on POCeHeparin showed increased expression of a-actin and decreased cell proliferation. This approach
can be easily adapted to modify other blood contacting devices such as stents where antithrombogenicity
and improved endothelialization are desirable properties.
� 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Cardiovascular disease is a leading cause of death and morbidity
in developed countries and patients diagnosed with this disease
often require revascularization ...
This study investigated the effects of red wine consumption on endothelial progenitor cells. The main findings were:
1) Drinking red wine for 3 weeks significantly increased circulating endothelial progenitor cell levels and improved endothelial function in healthy subjects.
2) Red wine ingestion enhanced nitric oxide bioavailability by increasing nitric oxide levels and decreasing asymmetrical dimethylarginine levels.
3) In vitro, red wine increased endothelial progenitor cell number, proliferation and function by modifying nitric oxide levels. It attenuated endothelial progenitor cell senescence and improved tube formation.
So in summary, moderate red wine consumption enhances circulating endothelial progenitor cells and improves their function through increasing nitric oxide bioavailability, which may contribute to the cardiovascular protective effects of
Several studies have shown that storing vascular grafts in saline or blood leads to endothelial cell damage. This damage contributes to graft failure after coronary artery bypass and peripheral artery bypass surgeries. Compounding pharmacies could create customized flushing and storage solutions for grafts that include nitric oxide substrates, antioxidants, and nutrients to support endothelial cell viability and function. Maintaining the health of the endothelial cells lining grafts using an optimal storage medium could improve long-term graft patency outcomes.
Effect of stemregenin1 and sb431542 small molecules on ex vivo expansion of u...Liberty University (LU)
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The document discusses body fluid compartments and their compositions. It notes that total body water makes up 60% of body weight, with two thirds located intracellularly and one third extracellularly. The extracellular fluid consists of interstitial fluid and blood plasma. Key electrolytes like sodium, potassium, calcium, and chloride are discussed along with their concentrations in plasma, interstitial, and intracellular fluids. Various volume expanders used to increase blood volume are also described, including crystalloids like saline and lactated Ringer's, as well as colloids like albumin, dextrans, gelatin, hydroxyethyl starch, and polyvinylpyrrolidone. Their mechanisms of action, properties, uses,
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2. A chapter outline covering red blood cell, white blood cell, platelet, and coagulation disorders.
3. A review of hematology anatomy and physiology including the components of blood and their roles.
Principles of Dielectric Blood Coagulometry as a Comprehensive Coagulation TestMarc-Aurele Brun
1) Dielectric blood coagulometry (DBCM) measures changes in the dielectric permittivity of whole blood over time during coagulation. It is sensitive to erythrocyte aggregation and shape changes caused by fibrin formation and platelet contraction.
2) DBCM testing of spontaneous coagulation showed three distinct phases corresponding to rouleau formation, erythrocyte aggregation with early fibrin formation, and erythrocyte shape transformation within the fibrin network.
3) Control experiments using activators and inhibitors revealed DBCM can assess platelet function, fibrin polymerization, fibrinolysis, and the anticoagulant effect of heparin. Therefore, DBCM has potential for comprehensive coagulation testing.
Bosche et al. - A differential impact of lithium on endothelium-dependent but...Dr. Bert Bosche
This document summarizes a study that investigated the effects of lithium on endothelium-dependent and endothelium-independent vessel relaxation. The study found that at lower therapeutic lithium concentrations (0.4 mmol/L), acetylcholine-induced endothelium-dependent vessel relaxation was slightly increased. However, at higher therapeutic and supratherapeutic lithium concentrations (0.8-100 mmol/L), endothelium-dependent vessel relaxation diminished in a concentration-dependent manner. In contrast, endothelium-independent vasorelaxation remained unaltered at any lithium concentration tested. Therefore, lithium has opposing effects on the endothelium depending on concentration, improving endothelium-dependent functions at lower levels but impairing them at
This study investigated the effects of red wine consumption on endothelial progenitor cells. The main findings were:
1) Drinking red wine for 3 weeks significantly increased circulating endothelial progenitor cell levels and improved endothelial function in healthy subjects.
2) Red wine ingestion enhanced nitric oxide bioavailability by increasing nitric oxide levels and decreasing asymmetrical dimethylarginine levels.
3) In vitro, red wine increased endothelial progenitor cell number, proliferation and function by modifying nitric oxide levels. It attenuated endothelial progenitor cell senescence and improved tube formation.
So in summary, moderate red wine consumption enhances circulating endothelial progenitor cells and improves their function through increasing nitric oxide bioavailability, which may contribute to the cardiovascular protective effects of
Several studies have shown that storing vascular grafts in saline or blood leads to endothelial cell damage. This damage contributes to graft failure after coronary artery bypass and peripheral artery bypass surgeries. Compounding pharmacies could create customized flushing and storage solutions for grafts that include nitric oxide substrates, antioxidants, and nutrients to support endothelial cell viability and function. Maintaining the health of the endothelial cells lining grafts using an optimal storage medium could improve long-term graft patency outcomes.
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The document discusses hematologic disorders and includes:
1. Objectives about defining terms, describing anatomy/physiology of the hematologic system, and pathophysiology of conditions like sickle cell disease.
2. A chapter outline covering red blood cell, white blood cell, platelet, and coagulation disorders.
3. A review of hematology anatomy and physiology including the components of blood and their roles.
Principles of Dielectric Blood Coagulometry as a Comprehensive Coagulation TestMarc-Aurele Brun
1) Dielectric blood coagulometry (DBCM) measures changes in the dielectric permittivity of whole blood over time during coagulation. It is sensitive to erythrocyte aggregation and shape changes caused by fibrin formation and platelet contraction.
2) DBCM testing of spontaneous coagulation showed three distinct phases corresponding to rouleau formation, erythrocyte aggregation with early fibrin formation, and erythrocyte shape transformation within the fibrin network.
3) Control experiments using activators and inhibitors revealed DBCM can assess platelet function, fibrin polymerization, fibrinolysis, and the anticoagulant effect of heparin. Therefore, DBCM has potential for comprehensive coagulation testing.
Bosche et al. - A differential impact of lithium on endothelium-dependent but...Dr. Bert Bosche
This document summarizes a study that investigated the effects of lithium on endothelium-dependent and endothelium-independent vessel relaxation. The study found that at lower therapeutic lithium concentrations (0.4 mmol/L), acetylcholine-induced endothelium-dependent vessel relaxation was slightly increased. However, at higher therapeutic and supratherapeutic lithium concentrations (0.8-100 mmol/L), endothelium-dependent vessel relaxation diminished in a concentration-dependent manner. In contrast, endothelium-independent vasorelaxation remained unaltered at any lithium concentration tested. Therefore, lithium has opposing effects on the endothelium depending on concentration, improving endothelium-dependent functions at lower levels but impairing them at
This document summarizes a review article about hydroxyethyl starch (HES), a synthetic colloid plasma volume expander commonly used in human and veterinary medicine. The summary discusses:
1) HES pharmacokinetics including concentration, molecular weight, molar substitution, and C2/C6 ratio which determine its volume expanding effects and breakdown in the body.
2) Reported benefits of HES include increasing intravascular volume and colloid osmotic pressure, but controversies exist regarding risks of acute kidney injury and coagulation issues from some HES products in human studies.
3) There is little data on HES use in veterinary medicine, and effects seen in human studies may not directly
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- Types of fluids used for resuscitation including crystalloids, colloids, blood, and hypertonic saline.
- Factors to consider in fluid resuscitation for hemorrhagic shock such as volume, blood oxygen carrying capacity, and coagulation status.
- Debate around delayed versus immediate fluid resuscitation for penetrating trauma, with some evidence that delayed resuscitation may improve outcomes.
- Uncertainties remain regarding best practices for fluid resuscitation depending on trauma type and patient characteristics.
1) The document discusses several studies that examine new pharmacological strategies and surgery for treating atherosclerotic conditions. It summarizes findings that calcium deposits play a role in atherosclerosis and can be quantified.
2) One study found that a combination therapy of EDTA, tetracycline, and supplements (ComET) led to decreases in coronary artery calcium scores for 57% of patients with coronary artery disease, implying regression of calcified plaque.
3) The TACT trial found that chelation therapy with EDTA reduced cardiovascular events by 18% overall and by 41% for patients with diabetes, showing potential benefit of removing heavy metals through chelation.
A recent publication by Dr. Sachin Kadam, CTO of Advancells, sheds light on natural osteoinductive bio-compatible scaffolds for effective clinical applications on musculoskeletal disorders. These scaffolds can enhance the efficiency of stem cell transplantation, and thus improve healthcare manifold.
1) Red blood cells undergo numerous biochemical changes during storage known as the "storage lesion" that may impair their function. These include loss of nitric oxide, potassium leakage into plasma, decreased 2,3-DPG and pH.
2) Retrospective studies show patients receiving older stored blood have worse outcomes like increased mortality, morbidity and organ dysfunction.
3) A case study describes a patient who developed hyperkalemia and cardiac arrest after receiving very old stored blood, highlighting the risks of potassium leakage from aged red blood cells.
ABSTRACT- Coronary artery disease (CAD) is suspected as a leading cause of mortality in developed countries. Due
to cholesterol and fat deposit plaque is forming into the inner walls of the arteries of the heart, which leads to narrowing
of blood vessels of heart and reduce the blood flow rate into heart. Proprotein convertase subtilisin-like kexin type 9
(PCSK9) is one of the candidate gene that regulate lipoprotein retention pathway of CAD development. It is a newly
discovered serine protease that plays a key role in LDL-C homeostasis by mediating LDL receptor (LDLR). The LDL
receptor is breakdown through a post transcriptional mechanism and induces the production of very low-density
lipoprotein in the fasting state. The aim of this study was to investigate the frequency of single nucleotide
polymorphism (SNP) of PCSK9 gene of 155 CAD patients and 102 ages matched healthy controls. Serum lipids
including total cholesterol (TC), triglycerides (TG), HDL, LDL, and VLDL were analyzed. PCR-RFLP analysis was
carried out to genotype regions carrying Eam 1104I restriction site in the PCSK9. Gene considering significant
difference in serum TC, TG, HDL-C, LDL-C and VLDL-C levels (P<0.001, <0.0001) of patients and control samples.
In CAD patients, G allele frequency is less than A allele frequency. G allele is responsible for decreasing the
LDL: HDL ratio which shows evidence in having its protecting effect on the occurrence of CAD in West Bengal Population.
Key-words- CAD, PCSK9, SNP, Eam1104I, Polymorphism, West Bengal population
1. The document discusses the physiology of hepatic microcirculation including the dual blood supply from the portal vein and hepatic artery.
2. It describes techniques to study hepatic microcirculation such as radioactive microspheres and various imaging modalities.
3. Modes of microvascular inflammation and dysfunction in liver injury are explained involving Kupffer cell activation, leukocyte recruitment, and endothelial dysfunction.
This document summarizes a study investigating the effects of kinsenoside, a constituent of Anoectochilus formosanus, on liver damage induced by carbon tetrachloride (CCl4) in mice. The study found that kinsenoside inhibited the activation of Kupffer cells (liver macrophages) stimulated by lipopolysaccharide in vitro. It also protected the liver from CCl4-induced injury in mice by reducing liver enzyme levels and improving liver histology. This protection is likely due to kinsenoside's ability to suppress Kupffer cell activation, as evidenced by reduced markers of Kupffer cell activation.
This document describes a protocol for engineering cardiac tissue using perfusion bioreactor systems. Cardiac cells are seeded onto porous scaffolds and cultured in bioreactors with perfusion of culture medium, which provides oxygen to the cells and overcomes limitations of conventional static culture. Two approaches are discussed: interstitial flow through porous scaffolds and flow through channel arrays in scaffolds. Perfusion improves cell viability, density, and function compared to static culture and enables engineering of thicker cardiac constructs.
1) The study investigated the association between preoperative levels of EPA, DHA, and AA in plasma and red blood cells, and changes in inflammatory markers after open heart surgery.
2) They found the postoperative concentrations of TNF-β decreased and hs-CRP, IL-6, IL-8, IL-18, and IL-10 increased compared to preoperative levels.
3) Preoperative EPA and AA levels were associated with changes in both pro-inflammatory and anti-inflammatory mediators, suggesting a complex role in the postoperative inflammatory response.
Assessment Of Mems Blood Separation Techniquesbrandypearson
The document discusses various techniques for blood cell separation at the point-of-care, including conventional centrifugation methods and newer microfluidic approaches. Size-based separation techniques discussed include obstacle-based deterministic lateral displacement and successive sieving, while affinity-based techniques include fluorescence labeling, adhesion to antibody-coated surfaces, magnetophoresis using magnetic beads, and dielectrophoresis using non-uniform electric fields. Challenges include achieving sufficient specificity, efficiency, and scale for point-of-care applications.
Cirrhosis results from different mechanisms of liver injury that lead to necroinflammation and fibrogenesis; Patients
with liver cirrhosis often require liver transplantation but it is affected by many problems, including relative operative
damage, high costs, lack of donors, and risk of rejection. Currently studies are shown the Stem cell therapy has the
potential to provide a valuable adjunct to the management of disease, Stem cell should be the natural candidates to
provide a renewable source of cells for transplantation.
The main mechanism of stem cell therapy is that stem cell capacity to differentiate into any of the hundreds of distinct
cell types that comprise the human body. In addition to their potential in therapeutics can be used to study the earliest
stages of human development and disease modeling using human cells.
Keywords: Cell Therapy; Liver Cirrhosis; Stem Cell; Transplantation. limitlessly, and often play the principal role in
liver regeneration
This document discusses body fluid compartments and volume expanders. It begins by outlining the total body water, intracellular fluid, and extracellular fluid compartments in the body. It then discusses the properties of various volume expanders, including crystalloids like saline and colloids like albumin, dextrans, gelatin, hydroxyethyl starch, and polyvinylpyrrolidone. For each type of volume expander, the document provides details on their composition, mechanism of action, advantages, and disadvantages. The document emphasizes that colloids are generally better than crystalloids at expanding plasma volume for longer durations.
This document summarizes recent advances in extracorporeal therapies. It discusses developments like hemofiltration, hemoperfusion with polymyxin B to remove endotoxins, high cut-off membranes that can remove cytokines, and cytokine binding therapies. It also reviews applications of extracorporeal therapies in conditions like sepsis, congestive heart failure, acute neurological injury, and liver failure. Overall, extracorporeal therapies have expanded from a focus on small solute removal to also address inflammatory mediator removal and organ support.
Integrated strategies for allogenic blood saving in major elective surgery ...anemo_site
This document discusses an integrated strategy implemented at a hospital in Rome, Italy since 2008 to reduce allogeneic blood transfusions for major elective surgeries. The strategy includes patient evaluation and supplementation if needed, along with autologous blood collection through pre-surgical donation or peri-surgical collection. Analysis of results from 2010 found that autologous unit usage increased 2.2 times, units not stored increased 2.4 times, and allogeneic unit transfusions were reduced by 65%, allowing for fresher blood transfusions to improve patient outcomes. The integrated strategy was more effective than previous practices at the hospital in conserving allogeneic blood supplies and reducing transfusion-related risks for patients.
In a two- to three-page paper (excluding the title and reference pag.docxrock73
In a two- to three-page paper (excluding the title and reference pages), explain the purpose of an income statement and how it reflects the firm’s financial status. Include important points that an analyst would use in assessing the financial condition of the company. Also, analyze Ford Motor Company’s income statement from its
2012 Annual Report
.
Your paper must be formatted according to APA style, and must include citations and references for the text and at least two scholarly sources.
.
In a substantial paragraph respond to either one of the following qu.docxrock73
In a substantial paragraph respond to either one of the following questions:
1.) Choose one source of energy, explain its origins, how does it impact our Earth, and what effect does it have on our planet?
OR
2.) Explain, with details, how geology influences the distribution of natural resources.
NO MINIMUM WORD LENGTH REQUIRED.
.
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2) Reported benefits of HES include increasing intravascular volume and colloid osmotic pressure, but controversies exist regarding risks of acute kidney injury and coagulation issues from some HES products in human studies.
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Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neu...Gul Muneer
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This document discusses fluid therapy in emergency care, covering topics such as:
- Types of fluids used for resuscitation including crystalloids, colloids, blood, and hypertonic saline.
- Factors to consider in fluid resuscitation for hemorrhagic shock such as volume, blood oxygen carrying capacity, and coagulation status.
- Debate around delayed versus immediate fluid resuscitation for penetrating trauma, with some evidence that delayed resuscitation may improve outcomes.
- Uncertainties remain regarding best practices for fluid resuscitation depending on trauma type and patient characteristics.
1) The document discusses several studies that examine new pharmacological strategies and surgery for treating atherosclerotic conditions. It summarizes findings that calcium deposits play a role in atherosclerosis and can be quantified.
2) One study found that a combination therapy of EDTA, tetracycline, and supplements (ComET) led to decreases in coronary artery calcium scores for 57% of patients with coronary artery disease, implying regression of calcified plaque.
3) The TACT trial found that chelation therapy with EDTA reduced cardiovascular events by 18% overall and by 41% for patients with diabetes, showing potential benefit of removing heavy metals through chelation.
A recent publication by Dr. Sachin Kadam, CTO of Advancells, sheds light on natural osteoinductive bio-compatible scaffolds for effective clinical applications on musculoskeletal disorders. These scaffolds can enhance the efficiency of stem cell transplantation, and thus improve healthcare manifold.
1) Red blood cells undergo numerous biochemical changes during storage known as the "storage lesion" that may impair their function. These include loss of nitric oxide, potassium leakage into plasma, decreased 2,3-DPG and pH.
2) Retrospective studies show patients receiving older stored blood have worse outcomes like increased mortality, morbidity and organ dysfunction.
3) A case study describes a patient who developed hyperkalemia and cardiac arrest after receiving very old stored blood, highlighting the risks of potassium leakage from aged red blood cells.
ABSTRACT- Coronary artery disease (CAD) is suspected as a leading cause of mortality in developed countries. Due
to cholesterol and fat deposit plaque is forming into the inner walls of the arteries of the heart, which leads to narrowing
of blood vessels of heart and reduce the blood flow rate into heart. Proprotein convertase subtilisin-like kexin type 9
(PCSK9) is one of the candidate gene that regulate lipoprotein retention pathway of CAD development. It is a newly
discovered serine protease that plays a key role in LDL-C homeostasis by mediating LDL receptor (LDLR). The LDL
receptor is breakdown through a post transcriptional mechanism and induces the production of very low-density
lipoprotein in the fasting state. The aim of this study was to investigate the frequency of single nucleotide
polymorphism (SNP) of PCSK9 gene of 155 CAD patients and 102 ages matched healthy controls. Serum lipids
including total cholesterol (TC), triglycerides (TG), HDL, LDL, and VLDL were analyzed. PCR-RFLP analysis was
carried out to genotype regions carrying Eam 1104I restriction site in the PCSK9. Gene considering significant
difference in serum TC, TG, HDL-C, LDL-C and VLDL-C levels (P<0.001, <0.0001) of patients and control samples.
In CAD patients, G allele frequency is less than A allele frequency. G allele is responsible for decreasing the
LDL: HDL ratio which shows evidence in having its protecting effect on the occurrence of CAD in West Bengal Population.
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1. The document discusses the physiology of hepatic microcirculation including the dual blood supply from the portal vein and hepatic artery.
2. It describes techniques to study hepatic microcirculation such as radioactive microspheres and various imaging modalities.
3. Modes of microvascular inflammation and dysfunction in liver injury are explained involving Kupffer cell activation, leukocyte recruitment, and endothelial dysfunction.
This document summarizes a study investigating the effects of kinsenoside, a constituent of Anoectochilus formosanus, on liver damage induced by carbon tetrachloride (CCl4) in mice. The study found that kinsenoside inhibited the activation of Kupffer cells (liver macrophages) stimulated by lipopolysaccharide in vitro. It also protected the liver from CCl4-induced injury in mice by reducing liver enzyme levels and improving liver histology. This protection is likely due to kinsenoside's ability to suppress Kupffer cell activation, as evidenced by reduced markers of Kupffer cell activation.
This document describes a protocol for engineering cardiac tissue using perfusion bioreactor systems. Cardiac cells are seeded onto porous scaffolds and cultured in bioreactors with perfusion of culture medium, which provides oxygen to the cells and overcomes limitations of conventional static culture. Two approaches are discussed: interstitial flow through porous scaffolds and flow through channel arrays in scaffolds. Perfusion improves cell viability, density, and function compared to static culture and enables engineering of thicker cardiac constructs.
1) The study investigated the association between preoperative levels of EPA, DHA, and AA in plasma and red blood cells, and changes in inflammatory markers after open heart surgery.
2) They found the postoperative concentrations of TNF-β decreased and hs-CRP, IL-6, IL-8, IL-18, and IL-10 increased compared to preoperative levels.
3) Preoperative EPA and AA levels were associated with changes in both pro-inflammatory and anti-inflammatory mediators, suggesting a complex role in the postoperative inflammatory response.
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The document discusses various techniques for blood cell separation at the point-of-care, including conventional centrifugation methods and newer microfluidic approaches. Size-based separation techniques discussed include obstacle-based deterministic lateral displacement and successive sieving, while affinity-based techniques include fluorescence labeling, adhesion to antibody-coated surfaces, magnetophoresis using magnetic beads, and dielectrophoresis using non-uniform electric fields. Challenges include achieving sufficient specificity, efficiency, and scale for point-of-care applications.
Cirrhosis results from different mechanisms of liver injury that lead to necroinflammation and fibrogenesis; Patients
with liver cirrhosis often require liver transplantation but it is affected by many problems, including relative operative
damage, high costs, lack of donors, and risk of rejection. Currently studies are shown the Stem cell therapy has the
potential to provide a valuable adjunct to the management of disease, Stem cell should be the natural candidates to
provide a renewable source of cells for transplantation.
The main mechanism of stem cell therapy is that stem cell capacity to differentiate into any of the hundreds of distinct
cell types that comprise the human body. In addition to their potential in therapeutics can be used to study the earliest
stages of human development and disease modeling using human cells.
Keywords: Cell Therapy; Liver Cirrhosis; Stem Cell; Transplantation. limitlessly, and often play the principal role in
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This document discusses body fluid compartments and volume expanders. It begins by outlining the total body water, intracellular fluid, and extracellular fluid compartments in the body. It then discusses the properties of various volume expanders, including crystalloids like saline and colloids like albumin, dextrans, gelatin, hydroxyethyl starch, and polyvinylpyrrolidone. For each type of volume expander, the document provides details on their composition, mechanism of action, advantages, and disadvantages. The document emphasizes that colloids are generally better than crystalloids at expanding plasma volume for longer durations.
This document summarizes recent advances in extracorporeal therapies. It discusses developments like hemofiltration, hemoperfusion with polymyxin B to remove endotoxins, high cut-off membranes that can remove cytokines, and cytokine binding therapies. It also reviews applications of extracorporeal therapies in conditions like sepsis, congestive heart failure, acute neurological injury, and liver failure. Overall, extracorporeal therapies have expanded from a focus on small solute removal to also address inflammatory mediator removal and organ support.
Integrated strategies for allogenic blood saving in major elective surgery ...anemo_site
This document discusses an integrated strategy implemented at a hospital in Rome, Italy since 2008 to reduce allogeneic blood transfusions for major elective surgeries. The strategy includes patient evaluation and supplementation if needed, along with autologous blood collection through pre-surgical donation or peri-surgical collection. Analysis of results from 2010 found that autologous unit usage increased 2.2 times, units not stored increased 2.4 times, and allogeneic unit transfusions were reduced by 65%, allowing for fresher blood transfusions to improve patient outcomes. The integrated strategy was more effective than previous practices at the hospital in conserving allogeneic blood supplies and reducing transfusion-related risks for patients.
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In a two- to three-page paper (excluding the title and reference pag.docxrock73
In a two- to three-page paper (excluding the title and reference pages), explain the purpose of an income statement and how it reflects the firm’s financial status. Include important points that an analyst would use in assessing the financial condition of the company. Also, analyze Ford Motor Company’s income statement from its
2012 Annual Report
.
Your paper must be formatted according to APA style, and must include citations and references for the text and at least two scholarly sources.
.
In a substantial paragraph respond to either one of the following qu.docxrock73
In a substantial paragraph respond to either one of the following questions:
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OR
2.) Explain, with details, how geology influences the distribution of natural resources.
NO MINIMUM WORD LENGTH REQUIRED.
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In a study by Dr. Sandra Levitsky, she considers why the economic, physical, and emotional challenges of providing chronic care for a family member have not produced more salient political demands for aggressive policy intervention (Hudson, 2014).
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References
Hudson, R. (Ed). (2014).
The new politics of old age policy
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.
in a minimum of 1000 words, describe why baseball is Americas past .docxrock73
in a minimum of 1000 words, describe why baseball is America's past time. As part of your paper you can share some of your memories of baseball. How did baseball mirror society(good and bad?) as a reflection of American society. Be sure to cite all of your sources and you must show direct evidence of integrating your textbook once per chapter as part of your final exam. Your paper should at include at least one resource from the library.
.
In a minimum 200 word response, describe some ways how the public .docxrock73
In a minimum 200 word response, describe some ways how the public has responded to the October 2001 USA Patriot Act. Has the public’s response been positive or negative? What are some pros and cons of the USA Patriot Act with the American public? Explain your answer.
Dempsey, J. S., & Forst, L. S. (2011, Pg. 213-214).
Police
. Clifton Park, NY: Delmar.
.
In a weekly coordination meeting, several senior investigators from .docxrock73
Senior investigators from a state crime lab requested that AB Investigative Services create standard operating procedures for processing computer evidence, as recent investigators have not properly understood how computer data works and technical issues related to evidence processing. ABIS was asked to provide 4 general guidelines for processing evidence to ensure investigators follow standard procedures.
In a memo, describe 1) the form and style of art as well as 2) the e.docxrock73
In a memo, describe 1) the form and style of art as well as 2) the engineering phenomenon – a substantial paragraph for each. You will need to research both the art and engineering, so each section of the memo should include citations from credible sources.
i need to wrote two paragraph also incloude two citation for each one
.
In a minimum 200 word response explain the problems that law enforce.docxrock73
In a minimum 200 word response explain the problems that law enforcement officials have faced regarding the issues of federal, state, and local jurisdictions attempting to intervene in tribal policing. How has this issue contributed to confusion and discontent with law enforcement? Dempsey, J. S., & Forst, L. S. (2011, Pg. 22-25). Police. Clifton Park, NY: Delmar.
.
In a minimum 200 word response explain some of the reasons why, in.docxrock73
In a minimum 200 word response explain some of the reasons why, in the context of span of control, it is more beneficial to
limit the number of officers reporting to one supervisor.
What factors can affect how many employees are supervised at one time?
Dempsey, J. S., & Forst, L. S. (2011, Pg.
Pg. 35-40
).
Police
. Clifton Park, NY: Delmar.
.
In a maximum of 750 words, you are required to1. Summarize the ar.docxrock73
In a maximum of 750 words, you are required to:
1. Summarize the article (include all necessary background information);
2. Identify, discuss and analyze the main issue covered in the article, making links to all secondary
issues, theories and concepts;
3. Critique the actions taken by management and the union, (i.e., what did each do particularly
well or poorly); and
4. Discuss how the event in the article affects the lives of people other than those in management
or the union
.
in a two- to- three page paper (not including the title and referenc.docxrock73
in a two- to- three page paper (not including the title and reference pages), explain how Foreign Direct Investment (FDI) would cause an increase in the BRIC (Brazil, Russia, India, and China) countries’ Gross Domestic Product (GDP).
Your paper must be formatted according to APA Style and include at least two scholarly sources to support your assertions.
.
In a two- to three-page paper (not including the title and reference.docxrock73
A balance sheet shows a company's financial position at a point in time by listing its assets, liabilities, and shareholders' equity. It reflects a company's financial status by indicating what it owns, owes, and the portion owned by shareholders. An analysis of Ford Motor Company's 2012 balance sheet from its Annual Report shows its assets, liabilities, and shareholders' equity at the end of 2012.
In a group, take a look at the two student essays included in this f.docxrock73
In a group, take a look at the two student essays included in this folder. For each of these essays: (1) outline the organization of the components, (2) label the components, (3) name the audience and purpose, (4) decide if you found the organization of the components to be effective, and if the components themselves were well written or poorly written. You'll type your notes into a Microsoft Word document, include the names of all group members, and then upload the document individually to your own iLearn dropbox.
.
BASEBALLRuns Scored (X)Wins (Y)7086987590654797048078795730716678661963867976457455667707918559674381731946418965471735797357361556
Develop a position paper on best practices for teaching English Learners. This paper should contain the student’s personal beliefs about and the best models to practice. Statements must be supported with research data. There must be at least THREE references. The textbook may serve as ONE reference (Education English Learners for a Transformed World) The paper must be typed using APA style, double spaced, and with a title page and a reference page. The paper should be no less than three pages in length.
The positon paper: why two way is the best method in Bilingual Education
1) Please explain the components of the Prism Model and why these components are important in creating a welcoming school that promotes success for English Learners.
2) There have been many programs and ideas in the US Public schools for how best to serve English Learners and close the gap between those who enter school speaking English and those who have to learn English along the way.
Following is a list of Bilingual Education Models that have been tried. According to the text book and the research of Virginia Collier and Wayne Thomas, please rate the following programs from 1-6 with 1 being the most effective program for student success and 6 being the least effective program for learning English:
__________Maintenance Bilingual Ed., Self-Contained
__________Transitional Bilingual Ed.
__________One-way Dual Language
__________Pull-out Bilingual Ed.
__________Two-way Dual Language
__________Enrichment Bilingual Education (30 min. per day)
The following programs are designed for ELs who do not live in an area where bilingual ed. is available or do not qualify for bilingual education due to the language they speak. Please rate the following ESL programs on a scale of 1-4 with 1 being the most successful way to teach English and 4 being the least effective program:
__________ESL Pull-out
__________Sheltered Instruction in the regular classroom
__________Total emersion with no language support
__________English enrichment, 30 minutes per day, by classroom teacher
3) Please explain the difference between a 50/50 model and a 90/10 model of Dual Language Education.
4) Why does 2-way Dual Language Education usually have better results than 1-way Dual Language Education?
5) In order to have an effective Dual Language program, there are two important things teachers should not do. What are they?
6) What does it mean to see other cultures not as a deficit but as a difference? Why is this idea important to your classroom?
7) We are required to have many formal assessments in our educational curriculum. However, informal assessment can be much more informative to the teacher of language learners. Please explain why Informal Assessments might be a better way for the teacher to know the true level of the student.
...
Based on Santa Clara University Ethics DialogueEthics .docxrock73
Based on Santa Clara University Ethics Dialogue
Ethics case studies
This is an extra credit assignment that I am offering for the first time this term. In this booklet, you will find 38 separate case studies. You are free to respond to any or all of these cases.
You may earn up to 5 extra credit points per question, based on the complexity of the case and the logic of your response. You may not earn more than 100 points (10 percent of your final grade).
You may find it helpful to read the paper “Four Tough Ethical Dilemmas” prior to responding.
While these are your opinions, citations are not expected; however, if you make use of the work of others, include APA style citations for complete credit.
Either cut and paste the cases you select to a separate file or use this file for your submission. If you use this file to submit a response, please delete those cases to which you are not responding.
Dr. Frick
Case 1: Family Loyalty vs. Meritocracy
A man was appointed president of the newly-acquired Philippine subsidiary of a large American company. He was reviewing the organization with the company's head of human resources. One thing the president noted was that the same names reoccurred frequently in several departments. "It is our tradition," commented the HR head. "Families take care of their own. If one family member gets a good job in a Philippine company, other members of the family apply to join that company and the first member there can help the whole family become successful by helping them get hired and by coaching them to be successful. The company benefits. Our costs of recruiting are lower, we know more about the people we hire, and the commitment to family success results in fewer performance and discipline problems because family members want to please their older relatives."
The president wondered how these practices would be regarded in a large American firm, and whether or not he should take action to change them.
1. Nepotism is not illegal, but is it ethical?
2. If the business is family-owned, does that make a difference?
3. How does national culture affect this discussion?
Case 2: Is the Two-Tier System Ethically Problematic
Employees at a cereal makers plant were “locked-out” from their jobs producing cereal for over 3 months. Company management and the union representing the employees reached a stalemate in negotiations resulting in the lockout. The union claims that the primary issue is the company’s demand of dramatically increasing the number of temporary workers, who would earn $6 less per hour and receive fewer benefits. Critics claim this effectively creates a two-tier system at the plant. Under the current agreement, the company may use temporary workers for up to 30% of the workforce, but the union claims the company is now pushing for 100%. The workers, who have had their health insurance suspended, fear that their jobs will either be replaced entirely by temporary workers, or they will be f ...
Barbara Corcoran Learns Her Heart’s True Desires In her.docxrock73
Barbara Corcoran Learns Her Heart’s True Desires
In her hilarious and lighthearted book, Shark Tales: How I Turned $1,000 Into a
Billion Dollar Business, Barbara Corcoran demonstrates the importance of knowing what
you really want out of life (Corcoran & Littlefield, 2011). As her title suggests, Barbara
founded her real estate company, The Corcoran Group, with only $1,000 and some big
dreams. Shortly after founding the company, Barbara took out a piece of paper and wrote
down some big goals for herself and the company. In 1978, she had only 14 sales agents
working for her, who earned a total of $250,000 in commissions. She set a goal of
doubling the number of agents and the commissions every year. So she put down 28 sales
people for 1979, 56 for 1980, and so on, all the way up to 1,792 salespeople in 1985 with
total commissions of $32,000,000. Barbara was amazed when she saw the fantastic sums
projected for 1985, and of course many people, when they see such amazing sums, would
dismiss the calculations as fantasy But as Barbara put it, she went to work the next day
hustling hard for her $32 million.
Real estate agents are paid largely by commission, which is about as close as you
can get to a pure form of contingent reward for performance. However, Barbara didn’t
rely solely on the commissions to motivate her workers. She threw theme parties and held
numerous social events to build a committed workforce. Good sales agents could always
move to another firm, but not every firm had Barbara’s positive attitude and fun-filled
atmosphere. In the early years of the firm, when money was tight, Barbara and her
relatives did the cooking for the outings and parties, and she found clever ways to
entertain people with skating parties and other lively activities. As the firm became larger
and more profitable, she even hired professional entertainers for the company’s midweek
picnics, which included elephant shows, daring rides on hot air balloons, horses, or
Harley Davidsons, etc. Barbara stated “I built my company on pure fun, and believe that
fun is the most underutilized motivational tool in business today. All of my best ideas
came when I was playing outside the office with the people I worked with” (Corcoran &
Littlefield, 2011, p. 283). What did she get in return for the fun atmosphere? She had the
“most profitable real estate company per person in the United States” (p. 284). By the
time she sold her agency in 2001, she had 1,000 agents working for her, and she had the
largest real estate agency in New York – clearly her motivational strategies attracted a
large number of productive employees.
Barbara Corcoran had sold her firm for $66 million. She thought that would make
her happy, but instead, it made her sad. Although she pretended to be happy with her new
wealth and freedom, she was “secretly miserable” (Corcoran & Littlefield, 2011, p. 232).
She had lost her purpose ...
This document provides context and summaries about Bapsi Sidhwa's novel Cracking India and Deepa Mehta's film adaptation Earth. It discusses the characters and plot of Earth, focusing on the abduction of Ayah. It analyzes themes in the novel like the child narrator, fallen women, masculinity, and the metaphor of India cracking. It also discusses the film adaptation and historical context of violence against women during the 1947 Partition of India and Pakistan, including government estimates of abductions.
Barriers of therapeutic relationshipThe therapeutic relations.docxrock73
Barriers of therapeutic relationship:
The therapeutic relationship between patient and nurse is often filled with barriers that can generate obstacles for the relationship and, in the end, the health system as a whole (Sfoggia et al.,2014). There are many factors that hinder building a therapeutic relationship: language, professional jargon, communication impairment, and cultural diversity (ibid).
Language:
Language can be an obstacle to nurse-patient communication because a patient may not be able to speak the same language and therefore communication is not possible (Levin,2006). The best way to overcome this barrier is providing a translator who can explain a professional facilitator's message easily to the patient(ibid). For instance, if the nurse only speaks English but the patient is only able to speak Arabic, a translation to the patient of what the professional facilitator is saying leads to less chance of misunderstanding (ibid). Translation also allows a patient to feel comfortable through being able to speak in their own language (ibid).
Medical jargon:
Jargon is a technical language that is comprehended by people in a specific industry or area of work (Leblanc et al.,2014). Health professionals often use jargon to communicate with each other(ibid). For example, T.B. disease stands for tubercle bacillus and HIV stands for human immunodeficiency virus (Mccrary & Christensen,1993). Jargon often makes sense to health professionals but a patient who does not understand these acronyms will not understand such communication, leading to a barrier in therapeutic relationship between patient and health professional (Leblanc et al.,2014).
Communication impairment:
Patients with communication impairment such as blindness, deafness and speech impairment often feel isolated, frustrated and self-conscious (O’Halloran et al.,2009). Some patients are born with such disabilities or have developed them as a result of disease (ibid). Therefore, nurses should provide enough time in order to describe any issue to such patients so that they do not feel uncomfortable or censured by health professionals, who must remain impartial (ibid).
Cultural diversity:
Patients often have various differences (Leblanc et al.,2014).Some of these differences are due to a patient's illness, social status, economic class, education and personality(ibid). However, according to Kirkham (1998), the deepest differences might be cultural diversity. Beheri (2009) points out that many nurses believe if they just treat patients with respect, they will avoid most cultural issues. Nevertheless, avoiding misunderstanding can be achieved through some knowledge of cultural customs, which might help and enable nurses to provide better health care to patients (ibid).
Facilitators of therapeutic relationship:
UNCRPD (2006) states that the most fundamental human right in hospital is communication. Patients are required to be provided with an effective communication method by nurs ...
Barada 2Mohamad BaradaProfessor Andrew DurdinReligions of .docxrock73
Barada 2
Mohamad Barada
Professor Andrew Durdin
Religions of the World Hum 201-02
March 23rd, 2018
References:
1. Rachel. Rachel’s Musings: Buddhism is a Religion. Retrieved from https://www.rabe.org/thoughts-on-buddhism/buddhism-is-a-religion/
2. Winfield, Pamela. The Conversation: Why so many Americans think Buddhism is just a philosophy. Retrieved from https://theconversation.com/why-so-many-americans-think-buddhism-is-just-a-philosophy-89488
Critical Analysis of the religious nature of Buddhism
The religious community often debates on whether Buddhism is categorized as a religion or as philosophical teaching. The answer to the question varies depending on an individual’s point of view. There are three main types of Buddhism practices across the world with each of them having smaller branches with slights variances in their teachings and beliefs. The different styles of Buddhist mainly encompass Theravada Buddhism, Vajrayana Buddhism, and Mahayana Buddhism. The various forms often have deities that are worshipped while others do not. Some often have scriptures while others do not usually believe in any physical form of the Buddhist teachings. The first article is authored by Rachel, a blogger, presenting the argument that Buddhism is a religion (Rachel, 1). On the other hand, the second article authored by Pamela Winfield recognizes Buddhism as a philosophy. Analyzing and comparing the two pieces having divergent views on the religious nature of Buddhism is crucial for understanding whether it is a religion or philosophy.
Summary of the articles
Rachel in her article considers Buddhism as a religion. The author acknowledges the fact that Mahayana Buddhism which is often found in greater part of Asia that includes Japan, Korea, and China often teaches on attaining enlightenment (Rachel, 1). The Mahayana often accept that every individual wishes to ensure the effective attainment of enlightenment and thus end the cycle of rebirth which others recognize as “Karma.” The article proceeds to state that Buddha is the greatest of the deities but is not worshipped. Instead, Buddha often inspires all those who practice doing as he once did. The author states that Buddhism often requires that the individuals that choose the wrong path attempt to re-accomplish these tasks in their next life alongside other punishments imposed on them by karma. The characteristics of this type of Buddhism thus often play a significant role in showing the religious nature of Buddhism. The author concludes by stating that Buddhism often contains all the different elements of a religion. Moreover, the article associates Buddhism with fallacies that characterize other religions and just as dangerous as other religions as well. A quote proves the claim on the dangerous nature of Buddhism that the author uses to summarize the teachings of Buddhism.
On the other hand, Winfield tends to focus on enlightening the readers on some of the aspects of Buddhism that ensures its a ...
Chapter wise All Notes of First year Basic Civil Engineering.pptxDenish Jangid
Chapter wise All Notes of First year Basic Civil Engineering
Syllabus
Chapter-1
Introduction to objective, scope and outcome the subject
Chapter 2
Introduction: Scope and Specialization of Civil Engineering, Role of civil Engineer in Society, Impact of infrastructural development on economy of country.
Chapter 3
Surveying: Object Principles & Types of Surveying; Site Plans, Plans & Maps; Scales & Unit of different Measurements.
Linear Measurements: Instruments used. Linear Measurement by Tape, Ranging out Survey Lines and overcoming Obstructions; Measurements on sloping ground; Tape corrections, conventional symbols. Angular Measurements: Instruments used; Introduction to Compass Surveying, Bearings and Longitude & Latitude of a Line, Introduction to total station.
Levelling: Instrument used Object of levelling, Methods of levelling in brief, and Contour maps.
Chapter 4
Buildings: Selection of site for Buildings, Layout of Building Plan, Types of buildings, Plinth area, carpet area, floor space index, Introduction to building byelaws, concept of sun light & ventilation. Components of Buildings & their functions, Basic concept of R.C.C., Introduction to types of foundation
Chapter 5
Transportation: Introduction to Transportation Engineering; Traffic and Road Safety: Types and Characteristics of Various Modes of Transportation; Various Road Traffic Signs, Causes of Accidents and Road Safety Measures.
Chapter 6
Environmental Engineering: Environmental Pollution, Environmental Acts and Regulations, Functional Concepts of Ecology, Basics of Species, Biodiversity, Ecosystem, Hydrological Cycle; Chemical Cycles: Carbon, Nitrogen & Phosphorus; Energy Flow in Ecosystems.
Water Pollution: Water Quality standards, Introduction to Treatment & Disposal of Waste Water. Reuse and Saving of Water, Rain Water Harvesting. Solid Waste Management: Classification of Solid Waste, Collection, Transportation and Disposal of Solid. Recycling of Solid Waste: Energy Recovery, Sanitary Landfill, On-Site Sanitation. Air & Noise Pollution: Primary and Secondary air pollutants, Harmful effects of Air Pollution, Control of Air Pollution. . Noise Pollution Harmful Effects of noise pollution, control of noise pollution, Global warming & Climate Change, Ozone depletion, Greenhouse effect
Text Books:
1. Palancharmy, Basic Civil Engineering, McGraw Hill publishers.
2. Satheesh Gopi, Basic Civil Engineering, Pearson Publishers.
3. Ketki Rangwala Dalal, Essentials of Civil Engineering, Charotar Publishing House.
4. BCP, Surveying volume 1
A review of the growth of the Israel Genealogy Research Association Database Collection for the last 12 months. Our collection is now passed the 3 million mark and still growing. See which archives have contributed the most. See the different types of records we have, and which years have had records added. You can also see what we have for the future.
Reimagining Your Library Space: How to Increase the Vibes in Your Library No ...Diana Rendina
Librarians are leading the way in creating future-ready citizens – now we need to update our spaces to match. In this session, attendees will get inspiration for transforming their library spaces. You’ll learn how to survey students and patrons, create a focus group, and use design thinking to brainstorm ideas for your space. We’ll discuss budget friendly ways to change your space as well as how to find funding. No matter where you’re at, you’ll find ideas for reimagining your space in this session.
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UPRAHUL
This Dissertation explores the particular circumstances of Mirzapur, a region located in the
core of India. Mirzapur, with its varied terrains and abundant biodiversity, offers an optimal
environment for investigating the changes in vegetation cover dynamics. Our study utilizes
advanced technologies such as GIS (Geographic Information Systems) and Remote sensing to
analyze the transformations that have taken place over the course of a decade.
The complex relationship between human activities and the environment has been the focus
of extensive research and worry. As the global community grapples with swift urbanization,
population expansion, and economic progress, the effects on natural ecosystems are becoming
more evident. A crucial element of this impact is the alteration of vegetation cover, which plays a
significant role in maintaining the ecological equilibrium of our planet.Land serves as the foundation for all human activities and provides the necessary materials for
these activities. As the most crucial natural resource, its utilization by humans results in different
'Land uses,' which are determined by both human activities and the physical characteristics of the
land.
The utilization of land is impacted by human needs and environmental factors. In countries
like India, rapid population growth and the emphasis on extensive resource exploitation can lead
to significant land degradation, adversely affecting the region's land cover.
Therefore, human intervention has significantly influenced land use patterns over many
centuries, evolving its structure over time and space. In the present era, these changes have
accelerated due to factors such as agriculture and urbanization. Information regarding land use and
cover is essential for various planning and management tasks related to the Earth's surface,
providing crucial environmental data for scientific, resource management, policy purposes, and
diverse human activities.
Accurate understanding of land use and cover is imperative for the development planning
of any area. Consequently, a wide range of professionals, including earth system scientists, land
and water managers, and urban planners, are interested in obtaining data on land use and cover
changes, conversion trends, and other related patterns. The spatial dimensions of land use and
cover support policymakers and scientists in making well-informed decisions, as alterations in
these patterns indicate shifts in economic and social conditions. Monitoring such changes with the
help of Advanced technologies like Remote Sensing and Geographic Information Systems is
crucial for coordinated efforts across different administrative levels. Advanced technologies like
Remote Sensing and Geographic Information Systems
9
Changes in vegetation cover refer to variations in the distribution, composition, and overall
structure of plant communities across different temporal and spatial scales. These changes can
occur natural.
Strategies for Effective Upskilling is a presentation by Chinwendu Peace in a Your Skill Boost Masterclass organisation by the Excellence Foundation for South Sudan on 08th and 09th June 2024 from 1 PM to 3 PM on each day.
How to Fix the Import Error in the Odoo 17Celine George
An import error occurs when a program fails to import a module or library, disrupting its execution. In languages like Python, this issue arises when the specified module cannot be found or accessed, hindering the program's functionality. Resolving import errors is crucial for maintaining smooth software operation and uninterrupted development processes.
हिंदी वर्णमाला पीपीटी, hindi alphabet PPT presentation, hindi varnamala PPT, Hindi Varnamala pdf, हिंदी स्वर, हिंदी व्यंजन, sikhiye hindi varnmala, dr. mulla adam ali, hindi language and literature, hindi alphabet with drawing, hindi alphabet pdf, hindi varnamala for childrens, hindi language, hindi varnamala practice for kids, https://www.drmullaadamali.com
Liberal Approach to the Study of Indian Politics.pdf
at SciVerse ScienceDirectBiomaterials 34 (2013) 30e41Con.docx
1. at SciVerse ScienceDirect
Biomaterials 34 (2013) 30e41
Contents lists available
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
The blood and vascular cell compatibility of heparin-modified
ePTFE vascular
grafts
Ryan A. Hoshi a, Robert Van Lith a, Michele C. Jen a,
Josephine B. Allen b, Karen A. Lapidos a,
Guillermo Ameer a,c,*
a Biomedical Engineering Department, Northwestern
University, Evanston, IL 60208, USA
b Material Science and Engineering Department, University of
Florida, Gainesville, FL 32611, USA
c Department of Surgery, Feinberg School of Medicine,
Chicago, IL 60611, USA
a r t i c l e i n f o
Article history:
Received 16 July 2012
Accepted 21 September 2012
Available online 12 October 2012
Keywords:
Vascular graft
Elastomer
Endothelial cell
Progenitor cell
2. Smooth muscle cell
Heparin
Hemocompatibility
Aminated poly(1,8-octanediol-co-citrate)
(POC)
* Corresponding author.
E-mail address: [email protected] (G. A
0142-9612/$ e see front matter � 2012 Elsevier Ltd.
http://dx.doi.org/10.1016/j.biomaterials.2012.09.046
a b s t r a c t
Prosthetic vascular grafts do not mimic the antithrombogenic
properties of native blood vessels and
therefore have higher rates of complications that involve
thrombosis and restenosis. We developed an
approach for grafting bioactive heparin, a potent anticoagulant
glycosaminoglycan, to the lumen of ePTFE
vascular grafts to improve their interactions with blood and
vascular cells. Heparin was bound to ami-
nated poly(1,8-octanediol-co-citrate) (POC) via its carboxyl
functional groups onto POC-modified ePTFE
grafts. The bioactivity and stability of the POC-immobilized
heparin (POCeHeparin) were characterized
via platelet adhesion and clotting assays. The effects of
POCeHeparin on the adhesion, viability and
phenotype of primary endothelial cells (EC), blood outgrowth
endothelial cells (BOECs) obtained from
endothelial progenitor cells (EPCs) isolated from human
peripheral blood, and smooth muscle cells were
also investigated. POCeHeparin grafts maintained bioactivity
under physiologically relevant conditions
in vitro for at least one month. Specifically, POCeHeparin-
coated ePTFE grafts significantly reduced
platelet adhesion and inhibited whole blood clotting kinetics.
POCeHeparin supported EC and BOEC
3. adhesion, viability, proliferation, NO production, and
expression of endothelial cell-specific markers von
Willebrand factor (vWF) and vascular endothelial-cadherin
(VE-cadherin). Smooth muscle cells cultured
on POCeHeparin showed increased expression of a-actin and
decreased cell proliferation. This approach
can be easily adapted to modify other blood contacting devices
such as stents where antithrombogenicity
and improved endothelialization are desirable properties.
� 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Cardiovascular disease is a leading cause of death and
morbidity
in developed countries and patients diagnosed with this disease
often require revascularization via bypass grafts to restore
blood
flow to tissue. Autologous vein bypass grafts, the gold-standard
of
care, cannot always be harvested, prompting the use of
prosthetic
vascular grafts. However, prosthetic vascular grafts have poor
long-
term patency, particularly when used in small diameter applica-
tions [1,2]. Therefore, there remains an urgent need for safe and
effective materials for the fabrication of vascular grafts.
Heparin and other anticoagulants have been incorporated into
biomaterials to inhibit intrinsic thrombogenicity [3e7]. Heparin
immobilization strategies have included physisorption, electro-
static deposition, and covalent bonding to surfaces [3,4,6,8,9].
For
meer).
All rights reserved.
4. example, cross-linked collagen surfaces have been used for the
covalent immobilization of heparin to improve blood
compatibility,
but the use of highly thrombogenic materials such as collagen
for
vascular grafts may be problematic [4,10]. Other strategies
include
the controlled release of heparin from electrospun materials but
soluble heparin can lead to increased risk of heparin-induced
thrombocytopenia, which can be fatal in some circumstances
[11]. To further augment vascular graft thromboresistance, some
promising approaches have combined heparin immobilization
with other known anti-clotting agents such as nitric oxide (NO)
or thrombomodulin, but these multi-factorial approaches are
complex and may be cost prohibitive for commercialization
[6,9].
Our lab has reported the synthesis of citric acid-based biomate-
rials for use in tissue engineering applications [12e19]. In
particular,
poly(1,8-octanediol-co-citrate) (POC) is an elastomeric
polyester
that can be used to form composites, fabricated to have micro
and
nano-architectures, and used as a coating to modify medical
devices
to potentially improve their performance [14,16e21]. In
previous
studies by our group and others, POC was demonstrated to be
mailto:[email protected]
www.sciencedirect.com/science/journal/01429612
http://www.elsevier.com/locate/biomaterials
http://dx.doi.org/10.1016/j.biomaterials.2012.09.046
http://dx.doi.org/10.1016/j.biomaterials.2012.09.046
http://dx.doi.org/10.1016/j.biomaterials.2012.09.046
5. R.A. Hoshi et al. / Biomaterials 34 (2013) 30e41 31
a biocompatible material with good hemocompatibility and
minimal acute and chronic inflammatory responses in vivo
[14,16,19,22]. POC also has been shown to support
endothelialization
under physiological flow conditions in vitro and in vivo [13,15].
Furthermore, the available carboxyl and hydroxyl groups on
POC
provide options to chemically modify the polymer network with
macromolecules to engineer new functionality.
The quest to develop a functional vascular graft would benefit
from a simple, effective and safe method to incorporate the
antithrombogenic and anticoagulant properties of heparin into
a biomaterial. In this regard, the effects of immobilized heparin
on
vascular cell processes should also be understood. Herein, we
describe the functionalization of POC with heparin and
investigate
the effects of POC-immobilized heparin on whole blood
clotting,
platelet adhesion, and the adhesion, viability, and phenotype of
endothelial cells and smooth muscle cells. As circulating
progenitor
cells can contribute to the endothelialization and arterial
healing
process, the effect of POCeHeparin on blood outgrowth
endothelial
cells (BOECs) is also investigated. BOECs are the progeny of
bone
marrow-derived circulating endothelial progenitor cells and
emerging evidence supports their use in cell-based therapies for
promoting postnatal vasculogenesis and clinical use in
cardiovas-
6. cular applications [23,24]. Differentiation of these cells may
promote the formation of a healthy endothelium on vascular
graft
surfaces [13,25,26]. This work also investigates whether
immobi-
lized heparin will have a negative effect on smooth muscle cell
proliferation.
2. Materials & methods
2.1. Synthesis and preparation of POC
All reagents and chemicals were purchased from SigmaeAldrich
(St. Louis, MO)
unless noted otherwise. The synthesis of POC has been
previously described [19].
Briefly, equimolar amounts of citric acid and 1,8-octanediol
monomers were melted
under a flow of nitrogen gas at 160e165 �C and then the
temperature of the system
was lowered to 140 �C to create a pre-polymer. Subsequently,
the pre-polymer was
purified in water, freeze-dried and then reconstituted in ethanol.
The pre-polymer
was post-polymerized in tissue culture polystyrene (TCP)
multiwell plates at
80 �C for 4 days. To fabricate POC-coated vascular grafts,
thin-walled ePTFE tubes
(6 mm inner diameter, 30e50 mm internodal distance, Zeus Inc.,
Orangeburg, SC)
were coated with 10% pre-polymer using a previously described
spin-shearing
method and post-polymerized at 80 �C for 4 days [13]. The
POC coating on the
ePTFE was verified visually using SEM and toluidine blue dye,
since it can bind with
7. high affinity to negatively charged carboxylic acid functional
groups [27].
2.2. Heparin immobilization to POC
POC samples were rinsed extensively in PBS at 37 �C for
several days to remove
unreacted monomers which may interfere with the conjugation
chemistry. Prior to
conjugation, POC-coated plates/vascular grafts were soaked in
0.1 M MES buffer (pH
5.6, containing 0.5 M NaCl) for 1 h. POC was covalently
modified using standard
carbodiimide chemistry. To covalently bind the carboxyl groups
of POC to the dia-
minohexane intermediate, a solution of 50 mM DH solution was
prepared with
300 mM N-(3 dimethylaminopropyl)-N0-ethylcarbodiimide
(EDC) and 150 mM
N-hydroxysuccinimide (NHS) catalysts in 0.1 M 2-(N-
morpholino) ethanesulfonic
acid (MES) buffer, pH 5.6, containing 0.5 M NaCl and
incubated with POC for 5 h at
room temperature. The POC was extensively rinsed with 2 M
NaCl and water to
remove excess reagents. The immobilized DH was conjugated to
the carboxylic acid
groups of heparin by incubating the POC with heparin solution
(2.5e5 mM) (heparin
sodium salt isolated from porcine intestinal mucosa, MW 15
kDa,167 units/mg) with
100 mM NHS and 200 mM EDC catalysts in MES buffer
overnight at room temper-
ature. A concentration of 5 mM heparin solution was determined
to be the maximal
solution concentration during conjugation as determined
8. previously by toluidine
blue assay (data not shown) and used for blood compatibility
and cell compatibility
studies [28]. Excess reagents and non-covalently bound heparin
were removed by
extensive washing with 2 M NaCl solution and water. For cell
compatibility studies,
samples were sterilized by exposure to ethylene oxide gas.
2.3. Immobilized heparin quantification and contact angle
measurements
The presence of heparin bonded to POC-coated ePTFE grafts
was detected and
quantified using the metachromatic dye, toluidine blue, as
described previously
[28]. The surface density of the POCeHeparin ePTFE coating
was evaluated for
stability after 14 days and 28 days of incubation in vitro at 37
�C in PBS and compared
against freshly prepared samples. To control for background dye
binding, the POCe
Heparin samples were compared against incubation-matched
controls consisting of
POC conjugated with diaminohexane. Static water-in-air contact
angle measure-
ments were taken over time using a custom-built contact angle
goniometer
(components from Rame-Hart, Inc., Mountain Lakes, NJ). A
total of three different
samples were measured for each surface type: ePTFE,
POCeePTFE and POCeHeparin
ePTFE over a 15-min period.
2.4. Heparin detection by X-ray photoelectron spectroscopy
(XPS)
9. Elemental analysis using X-ray photoelectron spectroscopy
(XPS) was per-
formed to verify the incorporation of diaminohexane and
heparin by confirming the
presence of nitrogen and sulfur peaks. POCeHeparin samples
were prepared using
2.5 mM heparin solution during conjugation. XPS spectra were
collected using
Omicron ESCALAB (Omicron, Taunusstein, Germany) with a
monochromated Al Ka
(1486.6 ev) 300 W X-ray source. Measurements consisted of
broad survey scans as
well as high-resolution N(1s) and S(1s) scans.
2.5. Isolation of blood outgrowth endothelial cells from human
peripheral blood
The isolation and characterization of BOECs obtained from
peripheral blood
have been previously described by our lab [13]. Briefly, forty-
five milliliters of
peripheral blood were collected from adult volunteers in the
presence of acid citrate
dextrose (ACD,
Solution
A; BD Biosciences). All procedures involving blood collec-
tion were performed in accordance with the regulations of the
Northwestern
University Institutional Review Board. Peripheral blood
10. mononuclear cells
(PB-MNCs) were isolated from whole blood via histopaque
density gradient
centrifugation using Accuspin tubes (Sigma Aldrich,
Milwaukee, WI). The isolated
PB-MNCs representing the starting populations were suspended
in endothelial cell
(EC) growth medium-2 (Lonza, Baltimore, MD) without the
fetal bovine serum
supplement but with 5% allogeneic human serum (Lonza). The
PB-MNCs were
seeded onto fibronectin-coated plates (BD Biosciences; San
Jose, CA), and cultured at
37 �C in a humidified incubator containing 5% CO2. After 4
days, non-adherent cells
were removed by complete media change, and media was
changed every 3e4 days
thereafter. BOECs were detected as tightly packed colonies with
the characteristic
cobblestone morphology of endothelial cells. After colony
isolation, BOECs were
expanded onto TCP and maintained through eight passages.
2.6. Bioactivity and antithrombogenicity assessment of
POCeHeparin ePTFE grafts
11. 2.6.1. Re-calcified whole blood clotting
The anti-clotting properties of POCeHeparin surfaces were
assessed using
modified re-calcified plasma and whole blood clotting assays
[13,15]. Whole blood
was collected from adult volunteers into ACD anticoagulant
(BD Biosciences,
Franklin Lakes, NJ). Sections of ePTFEePOCeHeparin grafts (5
mm in length) were
pre-weighed and placed in 1.5 mL centrifuge tubes. As a
control, non-modified
ePTFE and POC-coated ePTFE samples were used. The
anticoagulated whole blood
samples were re-calcified with the addition of 10% (v/v) 0.1 M
CaCl2 and then 750 mL
of re-calcified blood were then immediately incubated with
graft samples for 1 h at
room temperature. The grafts and all clotted blood were
carefully removed, briefly
blotted on a paper towel and weighed. The bioactivity of
POCeHeparin ePTFE
coating was evaluated after 14 days and 28 days of incubation
in vitro at 37 �C in PBS.
12. To evaluate the heparin bioactivity after adsorption with plasma
proteins, samples
were incubated with platelet poor plasma for 1 h at 37 �C prior
to the whole blood
clotting assay. Previous plasma protein studies have determined
that a majority of
protein becomes adsorbed to heparin-coated surfaces within this
time frame [29].
To account for the variability between blood donor collections,
each blood clotting
experiment included a set of non-modified ePTFE control
samples to normalize the
whole blood clot mass for POC and POCeHeparin experimental
samples.
2.6.2. Platelet adhesion
Whole blood was collected from adult volunteers into ACD
anticoagulant, which
has previously been reported to preserve platelet activity [13].
The blood was
centrifuged at 250 g for 15 min to obtain platelet-rich plasma
(PRP) supernatant. The
PRP preparation and platelet suspension buffer (PSB) used for
this experiment were
13. described previously [30]. Samples of ePTFE were cut into
disks using a cork borer to
match the surface area of 96 multiwell plates and gently pinned
down to remain in
place. PRP, diluted 1:10 in PSB, was incubated at 37 �C for 60
min with prepared
ePTFE samples and gently rinsed with warm PBS. The number
of adherent platelets
was determined by detecting the amount of lactate
dehydrogenase (LDH) present
after cell lysis as previously described [15]. Briefly, adherent
platelets were lysed by
incubation with 2% Triton-PSB buffer for 30 min at 37 �C. A
colorimetric substrate for
LDH (Roche Diagnostics Corporation, Indianapolis, IN) was
added and incubated for
20 min at 37 �C. The reaction was stopped with the addition of
1N hydrochloric acid.
The optical density was measured at 490 nm with a reference
wavelength of 650 nm.
A calibration curve was generated from a series of serial
dilutions of a known platelet
concentration and used to determine the number of adhered
platelets. The
14. Fig. 1. Detection of immobilized heparin by X-ray
photoelectron spectroscopy. XPS spectra for N(1s) and S(1s) for
POC, POC conjugated with DH (POCeDH), and POCeHeparin.
R.A. Hoshi et al. / Biomaterials 34 (2013) 30e4132
morphology of adhered platelets was assessed via scanning
electron microscopy
(SEM). Briefly, adherent platelets were fixed using 2.5%
glutaraldehyde in PBS for at
least 2 h, dehydrated in a graded series of ethanol, and freeze-
dried. The samples
were then sputter-coated with a 7-nm layer of gold and observed
using scanning
electron microscopy (SEM 3400N, Electron Probe
Instrumentation Center, North-
western University).
2.7. Effect of immobilized heparin on vascular cells
Human umbilical vein endothelial cells (HUVEC) (Lonza,
Baltimore, MD)
(passages 3e6) were cultured in EGM-2 media. Human aortic
smooth muscle cells
15. (HASMC) (Lonza) (passages 3e5) were cultured in SmGM-2
media. BOECs were
isolated and cultured as described earlier. All cells were
cultured at 37 �C in
a humidified incubator containing 5% CO2. HUVECs and
HASMCs (seeding density
1 � 104 cells/cm2) were seeded onto POC, POCeHeparin and
TCP surfaces. Similarly,
the seeding density for BOECs was 7.5 � 103 cells/cm2. Cell
culture media was
changed every 3 days.
2.7.1. Cell proliferation
At predetermined time intervals, cells were lysed and quantified
using a Pico-
Green DNA assay (Molecular Probes, Carlsbad, CA) and
compared against standards
of known cell numbers.
2.7.2. Cell viability
Cell viability of adherent cells was assessed using a live/dead
cell viability assay
kit (Invitrogen, Carlsbad, CA) after 4 days of culture. Following
16. manufacturer’s
instructions, after incubation with the live/dead staining
solution, the cells were
gently rinsed in warm PBS and adherent cells were imaged for
viability by fluo-
rescence microscopy.
2.7.3. Cell phenotype
Cells were fixed with 4% paraformaldehyde and blocked with
10% normal goat
serum. HUVECs and BOECs were probed with primary
antibodies to EC-specific
markers von Willebrand factor (vWF) (Dako Cytomation,
Carpenteria, CA) and
CD144 (VE-Cadherin) (R&D Systems, Minneapolis, MN).
HASMCs were probed with
Fig. 2. Schematic of the bioactive PO
a-actin smooth muscle (abCam, Cambridge, MA). Cells were
counterstained
with Hoechst and analyzed by fluorescence microscopy (Nikon
TE2000U). POC and
POCeHeparin surfaces have background fluorescence when
stained with Hoechst;
therefore, the background fluorescence was digitally removed
17. for clarity.
2.7.4. Nitric oxide production
HUVECs and BOECs were cultured on TCP, POC and
POCeHeparin surfaces for up
to 4 days and then probed for production of nitric oxide using
the fluorescent probe
5,6-diaminofluorescein diacetate (DAF-2 DA, Santa Cruz
Biotechnology, Santa
Cruz, CA) [31]. After cell detachment with trypsin-EDTA, cell
suspensions were
incubated with 5 mM DAF-2 DA for 1 h at 37 �C. The
fluorescence of DAF was excited
at 488 nm and emitted fluorescence was measured at 530/40 nm
using a BD LSR2
flow cytometer (BD Biosciences, San Jose, CA).
2.8. Statistical analysis
Numerical data are reported as mean � standard deviation (SD).
The statistical
significance between two sets of data was calculated using a
two-tail Student’s
t-test. One way and two way ANOVA tests were used to
18. measure differences for
experiments with multiple data sets with a post hoc Bonferroni
test performed
between groups with significant differences to correct for the
multiple pairwise
comparisons. A value of p < 0.05 was considered to be
statistically significant.
3. Results
3.1. Heparin immobilization to POC
The covalent modification of thin POC films with diaminohex-
ane and heparin was assessed by XPS for N1s and S1s spectra
(Fig. 1). The presence of nitrogen from diaminohexane amine
functional groups was detected for POCediaminohexane in addi-
tion to POCeHeparin surfaces. As expected, no nitrogen or
sulfur
peaks were detected for unmodified POC surfaces and no sulfur
CeHeparin ePTFE vascular graft.
R.A. Hoshi et al. / Biomaterials 34 (2013) 30e41 33
peaks were detected for POCeDH surfaces. Surfaces conjugated
19. with heparin showed peaks for sulfur.
3.2. Fabrication of the POCeHeparin ePTFE graft
Vascular grafts were prepared by coating ePTFE tubes with
a coating of POC using a “spin shear” coating technique
developed in
our lab [13]. The POC coating was used to prepare the ePTFE
for
subsequent covalent immobilization of heparin via a DH linker
molecule (Fig. 2). The POC pre-polymer is cross-linked within
the
node and fibril structure of the ePTFE lumen at 80 �C. The
POC and
POCeHeparin coatings are capable of modifying the ePTFE
without
significantlyaltering the original node and fibril architecture
(Fig. 3).
The presence of heparin on the luminal surface of modified
ePTFE grafts was confirmed visually by a purple color change
from
the toluidine blue stain (Fig. 3D). The cationic toluidine blue
dye can
also bind to negatively charged carboxyl groups present in POC
20. and
showed significant background staining for POC-coated ePTFE.
The
Fig. 3. Characterization of POCeHeparin vascular grafts. SEM
micrograph of the luminal surf
Arrows indicate areas with POC-coated fibrils (scale bars: 20
mm). (D) POCeHeparin coatin
change, en face preparations of graft segments show lumen side
up. (E) Heparin surface dens
28 days incubation in vitro at 37 �C in PBS, N.S. ¼ “not
significant”, n ¼ 6, mean � SD. (F) Sta
coated ePTFE grafts. *p < 0.05, significantly less than ePTFE,
**p < 0.01, significantly less than
[For interpretation of the references to colour in this figure
legend, the reader is referred t
purple color change was only seen on POCeHeparin ePTFE. No
dye
binding was observed for ePTFE control grafts. The toluidine
blue
dye was also used to quantify the surface density of
POCeHeparin-
coated ePTFE grafts and confirm the stability of the
immobilized
heparin after incubation under physiological conditions in vitro
(Fig. 3E). There were no statistically significant changes in the
21. heparin surface density over time (p ¼ 0.58). The heparin
surface
density after incubation for 14 days and 28 days was 35.5 �
11.1 and
37.4 � 8.9 ng/mm2, respectively, compared with 47.2 � 14.6
ng/
mm2 for freshly prepared samples.
Static water-in-air contact angle measurements also confirmed
the successful modification of the ePTFE luminal surface with
a coating of POC and surface immobilized heparin. After a 5-
min
time period, the POCeHeparin ePTFE had a significantly
reduced
water-in-air contact angle compared with ePTFE and
POCeePTFE
surfaces (Fig. 3F). After 15 min, ePTFE, POCeePTFE and POCe
Heparin ePTFE had water contact angles of 98.43 � 5.32�,
86.67 � 6.53� and 20 � 18.17�, respectively.
ace of (A) unmodified ePTFE, (B) POC-coated ePTFE and (C)
POCeHeparin-coated ePTFE.
g on ePTFE grafts was determined by toluidine blue staining
showing a purple color
ity on freshly prepared (Day 0) POCeHeparin-coated ePTFE
grafts and after 14 days and
22. tic water-in-air contact angle measurements for ePTFE,
POCeePTFE and POCeHeparin-
ePTFE and POC, ***p < 0.001 significantly less than ePTFE
and POC, n ¼ 4, mean � SD.
o the web version of this article.]
Fig. 5. POCeHeparin coating remains bioactive when exposed to
human plasma.
Whole blood clot mass for ePTFE and POCeHeparin grafts pre-
incubated with platelet
poor plasma as percent of ePTFE control surfaces, *p < 0.05
compared with ePTFE
control and ePTFE þplasma samples, n ¼ 4, mean � SD.
R.A. Hoshi et al. / Biomaterials 34 (2013) 30e4134
3.3. Bioactivity and antithrombogenicity assessment of POCe
Heparin ePTFE grafts
3.3.1. Re-calcified whole blood clotting
The bioactivity of POCeHeparin ePTFE grafts was assessed
over
23. a 28-day incubation in vitro at 37 �C in PBS. A re-calcified
whole
blood clotting assay demonstrated the potent anticoagulant
activity
of the POCeHeparin ePTFE grafts (Fig. 4). The POCeHeparin
grafts
had a dramatic effect upon whole blood clotting for all time
points
tested: Days 0, 14 and 28. Upon visual inspection, the
POCeHeparin
graft surface remained relatively clean compared with the POC-
coated and bare ePTFE grafts (Fig. 4A). The POCeHeparin
grafts
had significantly less blood clot formation compared with
ePTFE
and POCeePTFE grafts at all time points tested up to 28 days
(p < 0.05). In addition, the POCeHeparin grafts had no
significant
change in anti-clotting activity over the same time period. In
contrast, the POC-coated grafts were not significantly different
from ePTFE controls at all time points tested. At the 28-day
time
point, the POCeHeparin and POCeePTFE samples had blood
clot
formation that was 15.6 � 10.2% and 70.6 � 24.4% of ePTFE
24. controls,
respectively.
The POCeHeparin ePTFE samples were also evaluated for anti-
clotting activity after incubation in 100% platelet poor plasma
for
1 h at 37 �C. The POCeHeparin samples pre-incubated in
plasma
had significantly less whole blood clot formation which was
only
4.6 � 5.7% compared with ePTFE control grafts (p < 0.05) (Fig.
5).
Fig. 4. Bioactivity of POCeHeparin grafts over time. (A) Whole
blood clot formation on
ePTFE graft segments, POC, and POCeHeparin-coated ePTFE
graft segments after 28
days incubation in vitro at 37 �C in PBS. Left image panels
show graft lumen en face and
right image panels show graft cross-sections. (B) Whole blood
clot mass for POC and
POCeHeparin-coated ePTFE as percent of ePTFE control
surfaces, *p < 0.05, signifi-
cantly less than ePTFE and POC. N � 4, mean � SD. Note:
ePTFE control samples (N � 4)
were also included for each blood clotting experiment (time
25. points Day 0eDay 28) and
used to normalize whole blood clot mass for POC and
POCeHeparin samples.
3.3.2. Platelet adhesion
In addition to whole blood clotting, the grafts were evaluated
for
platelet adhesion using platelet-rich plasma diluted in platelet
suspension buffer (Fig. 6). Platelet-rich plasma contains
clotting
factors present in the plasma as well as proteins such as
fibrinogen,
contained within the a-granules of platelets, which are capable
promoting clot formation [32]. There were numerous adherent
and
spread platelets within a clot on the luminal surfaces of both
POC-
coated and ePTFE control grafts as visualized by SEM (Fig. 6A
and
B). The adherent platelets as seen by SEM are approximately
2.5 mm
in diameter which corresponds to average platelet size in
humans
and the adhered and spread platelet morphology is similar to
previously reported literature [33,34]. In comparison, there was
26. a dramatic difference with the POCeHeparin grafts which
remained
relatively pristine with the ePTFE node and fibril architecture
still
clearly visible (Fig. 6C). An LDH assay quantified the number
of
adherent platelets on the different vascular graft surfaces. The
number of adherent platelets on POCeHeparin ePTFE grafts was
significantly less than POC and ePTFE grafts (p < 0.05). The
number
of platelets on POC and ePTFE grafts was 4.5 � 107 � 3.5 �
106 and
4.8 � 107 � 5.8 � 106 per cm2, respectively. In contrast, the
number
of adherent platelets to POCeHeparin grafts was only
1.5 � 106 � 4.7 � 105 per cm2. The relatively small number of
adherent platelets on the POCeHeparin surface as visualized by
SEM
and quantified by the LDH assay, demonstrates the ability for
the
POCeHeparin surfaces to strongly inhibit platelet adhesion
when
challenged with a relatively high concentration of platelets
(platelet-
rich plasma).
27. 3.4. Effect of immobilized heparin on vascular cells
3.4.1. HUVEC and BOEC viability and proliferation on
POCeHeparin
HUVECs and BOECs exhibited good attachment, spreading, and
a high degree of viability on all surfaces tested (Fig. 7). In
addition,
HUVECs and BOECs stained positive for vWF and VE-Cadherin
(Fig. 8). POCeHeparin supported cell proliferation for both
endo-
thelial cell types although there was some inhibition of
HUVECs
proliferation on POCeHeparin (Fig. 9). Specifically, the
HUVEC
surface density after 4 days on POCeHeparin was 1.25 � 105
cells/
cm2 compared with 1.63 � 105 cells/cm2 and 1.50 � 105
cells/cm2
for TCP and POC surfaces, respectively. Although there were
Fig. 6. Effect of POCeHeparin on platelet adhesion. SEM
28. micrographs of samples after incubation in platelet-rich plasma:
(A) ePTFE, (B) POC-coated ePTFE and (C) POCeHeparin-
coated ePTFE. (D) Platelet adhesion quantified by LDH, *p <
0.05 compared with ePTFE and POC samples, n � 6, mean �
SD. (AeC) Scale bars: 50 mm.
Fig. 7. Cell viability of adherent cells on POCeHeparin.
HASMCs, BOECs and HUVECs on TCP, POC and POCeHeparin
surfaces after culturing for 4 days. Green: live cells, Red: dead
cells. Scale bars: 100 mm. [For interpretation of the references
to colour in this figure legend, the reader is referred to the web
version of this article.]
R.A. Hoshi et al. / Biomaterials 34 (2013) 30e41 35
R.A. Hoshi et al. / Biomaterials 34 (2013) 30e4136
significantly fewer HUVECs on the POCeHeparin surfaces after
4
days, the HUVECs were capable of forming a confluent cell
mono-
layer on the POCeHeparin surface after 7 days of culture (Fig.
9).
Additionally, HUVECs maintained expression of endothelial
29. cell
markers and exhibited no signs of decreased cell viability on
POCe
Heparin. In contrast, BOECs proliferated at the same rate on all
surfaces, although at a slower rate when compared to HUVECs.
The
BOEC surface density after 4 days on POCeHeparin was 1.12 �
104
cells/cm2 compared with 1.15 � 104 cells/cm2 and 1.27 � 104
cells/
cm2 for TCP and POC surfaces, respectively. The number of
BOECs
on the different surfaces after 4 days of culture was not
statistically
significant.
3.4.2. HASMC viability and proliferation on POCeHeparin
HASMCs showed good adhesion and viability on all surfaces
tested with minimal signs of dead/dying cells (Fig. 7). The
HASMCs
on POC and POCeHeparin also had a more elongated and less
spread morphology compared with HASMCs cultured on TCP.
Interestingly, HASMCs cultured on POC and POCeHeparin
surfaces
30. had greater a-actin expression, which is an indicator of a more
physiological contractile phenotype (Fig. 10B). For HASMC
prolif-
eration, there were significantly fewer cells on both POC and
POCe
Heparin surfaces after 4 days of culture (Fig. 10A). The
HASMC
surface density after 4 days on POCeHeparin was 7.39 � 104
cells/
cm2 compared with 1.32 � 105 cells/cm2 and 5.06 � 104
cells/cm2
for TCP and POC surfaces, respectively. The proliferation data
is in-
line with the a-actin staining, which demonstrates that POC and
POCeHeparin surfaces promote a more contractile and less
prolif-
erative smooth muscle cell phenotype.
Fig. 8. The effect POCeHeparin on endothelial cell phenotype.
(A) Immunofluorescence stain
100 mm). [For interpretation of the references to colour in this
figure legend, the reader is
3.4.3. Nitric oxide production
HUVECs and BOECs cultured on POC and POCeHeparin
surfaces
31. produced similar levels of nitric oxide as cells cultured on TCP
(Fig. 11). These results demonstrate the ability for
POCeHeparin
surfaces to support functional nitric oxide producing cells nor-
mally present in healthy endothelium.
4. Discussion
The quest for the ideal prosthetic vascular graft has generated
a significant amount of research on the development of novel
biomaterials and surface modification techniques to improve the
clinical outcome of bypass surgeries. Of the various strategies
investigated over the years, only endothelial cell-based and
heparin-based approaches have shown significant promise in
regards to patient outcome [35,36]. Nevertheless, more research
is
needed to overcome the challenges associated with the in vitro
or
in vivo endothelialization of prosthetic grafts to enable
widespread
clinical use.
With regard to heparin immobilization, non-covalent and cova-
lent strategies have been reported with the latter more desirable
to
32. minimize the release of heparin into the systemic circulation
[11,37].
Although a comprehensive review of all antithrombogenic
strategies
for cardiovascular biomaterials is beyond the scope of this
current
paper, Table 1 provides a summary of heparin-modification
strate-
gies and current technologies that are specific to the
development of
vascular graft biomaterials. A more detailed review of antith-
rombogenic coating technologies for vascular grafts has been
prepared by Tatterton et al., and Kapadia et al. [38,39]. In
recent
ing for HUVECs and BOECs, Red: vWF, Green: VE-Cadherin,
Blue: cell nuclei (scale bars:
referred to the web version of this article.]
R.A. Hoshi et al. / Biomaterials 34 (2013) 30e41 37
years, there has been a growth of commercially available
heparin-
modified vascular graft materials such as the Propaten� graft
(W.L. Gore & Associates, Inc.) and the Intergard� graft
33. (Maquet
Cardiovascular Inc.). However, the long-term performance and
benefit of such commercially available technologies remains to
be
answered as the Intergard� graft has demonstrated no
difference in
performance after 5 years compared with human umbilical vein
grafts and PTFE grafts [40,41]. Furthermore, the longest
prospective
and randomized clinical trial to date for the Propaten� graft is
only for 1 year, but results from this study reveal a decrease in
primary graft failure compared with PTFE as a bypass for lower
limb
ischemia [42].
The Carmeda Bioactive Surface Modification (CBAS�)
technique
is the basis for the Propaten� vascular graft manufactured by
W.L.
Gore, which is among the most widely used of commercially
available heparin-bonded vascular grafts [7,43]. CBAS� is
based on
layer-by-layer deposition of oppositely charged
polyelectrolytes,
followed by covalent attachment of heparin, via its reducing
34. end, to polyethyleneimine. The CBAS� technology requires
toxic
chemical reagents and numerous surface modification steps
involving nitrous acid-treated heparin and cross-linking of poly-
ethyleneimine layers with glutaraldehyde [7,44]. Previous work
has
shown that biomaterial surfaces prepared with
polyethyleneimine
by layer-by-layer deposition exhibit cytotoxicity and inhibit cell
proliferation [45].
In this study, we developed a new and easily implemented
approach to covalently link bioactive heparin to the lumen of
ePTFE
grafts using a thermally cross-linked POC elastomer and
immobili-
zation chemistry that targets the carboxyl groups on heparin. As
endothelial and smooth muscle cells play an important role in
ini-
timal hyperplasia and blood vessel homeostasis, the effects of
immobilized heparin on these cell types were also evaluated.
The
Fig. 9. HUVEC and BOEC cell growth on POCeHeparin. (A)
Cell surface density for HUVECs a
black bars ¼ POCeHeparin surfaces for all panels. #p < 0.01
35. compared with POC, **p < 0.001
of culture on POCeHeparin (scale bar: 100 mm).
POC copolymer chain is composed of a large number of
carboxyl and
hydroxyl functional groups that are amenable for a variety of
surface
functionalization strategies for tailoring surface chemistry
and/or
immobilizing bioactive molecules or peptides. The copolymer
can
be readily coated onto the nodes and fibrils of ePTFE vascular
grafts
(Fig. 3) without significantly altering vascular wall thickness,
mechanical properties, or ePTFE node/fibril microstructure
[16].
The stability of surface bound molecules for improving vascular
conduit blood contacting properties is crucial for long-term
vascular graft performance and patency. For example, large
amounts of eluted heparin can potentially become lethal due to
complications associated with heparin-induced
thrombocytopenia
[46]. The heparin surface density on POCeHeparin ePTFE grafts
(w36e46 ng/mm2) remained stable over a 28-day period in vitro
under physiological conditions with no significant change in
36. heparin surface density over time. In contrast, although heparin-
ized stainless steel stents have similarly reported values, they
experience considerable degradation (nearly 40%) after one
month [33]. Other previous work with heparinization of poly-
urethanes has shown a maximum heparin surface density of
approximately 11e23 ng/mm2 with stability evaluated after only
4
days [47e49].
It is important to evaluate the activity of immobilized heparin
because heparin surface density is not necessarily proportional
to
antithrombogenic activity [50,51]. Aside from factors such as
heparin molecular weight and purification methods which may
affect heparin activity, the type of covalent modification may
alter
accessibility of the heparin’s ATIII-binding site [50]. In this
report,
POCeHeparin-coated ePTFE grafts significantly inhibited whole
blood clotting and maintained long-term bioactivity in vitro for
up to one month. These results are promising because other
studies
have shown inconsistent immobilized heparin bioactivity after
nd BOECs on TCP, POC and POCeHeparin surfaces. White bars
¼ TCP, gray bars ¼ POC,
37. compared with TCP. n � 5, mean � SD. (B) Confluent
monolayer of HUVECs after 7 days
Fig. 10. The effect of POCeHeparin on smooth muscle cell
growth and phenotype. (A) Cell surface density for HASMCs on
TCP, POC and POCeHeparin surfaces. #p < 0.01 compared
with POC, **p < 0.001 compared with TCP. n � 5, mean � SD.
(B) Immunofluorescence staining for HASMCs, Green: a-actin,
Blue: Cell nuclei (scale bars: 100 mm). [For interpretation
of the references to colour in this figure legend, the reader is
referred to the web version of this article.]
R.A. Hoshi et al. / Biomaterials 34 (2013) 30e4138
much shorter time scales (w5 days) [52]. In this regard, the use
of
spacer molecules such as acrylamide has been shown to improve
the bioactivity of immobilized heparin [50]. In this work, the
dia-
minohexane linker likely allows for improved bioactivity due to
increased mobility of heparin’s ATIII-binding site. Activation
of ATIII
and subsequent inhibition of pro-clotting factors involved in
coagulation are key to reduced blood coagulation when blood
38. comes in contact with surfaces displaying immobilized heparin
Fig. 11. Nitric oxide production in HUVEC and BOEC cultured
on POCeHeparin. NO-posit
(B) cultured on TCP, POC and POCeHeparin surfaces. NO-
positive cells were compared rela
[53]. The anticoagulant property of immobilized heparin in the
grafts was also maintained after incubation with human plasma.
This characteristic is important because clotting factors present
in
the plasma may adsorb to a blood contacting surface leading
acti-
vation of the coagulation cascade [54]. It is believed that
surfaces
with immobilized heparin are capable of inhibiting the process
of
serum protein adsorption, activation and denaturation involved
in
thrombus formation [29,55].
ive cells were analyzed by flow cytometry using DAF-2 DA.
HUVECs (A) and BOECs
tive to “background” samples for which DAF-2 DA cell
treatment was omitted.
39. Table 1
Current technologies and methods for developing heparin-
modified vascular graft biomaterials.
Author/Company Heparin incorporation
technique/coating technology
Biomaterial Longest performance
time point measured
Outcome Clinically available
Lord et al. [3] Surface adsorption of perlecan
heparan sulfate proteoglycan
ePTFE vascular grafts 6 weeks in vivo
(ovine model)
Y platelet adhesion
in vitro (fresh samples),
improved EC coverage
and decreased platelet
adhesion in vivo
(qualitative analysis
only at 6 weeks)
40. Janairo et al. [66] EDC/NHS using
diamino-PEG linker
Electrospun PLLA
vascular graft
1 month in vivo
(rat model)
Heparin-modified
grafts had 86% patency
compared with
43e57% for control grafts.
Chuang and
Masters [49]
PEI modified polyurethane,
aldehyde activated heparin
Polyurethane films 5 days in vitro [ platelet adhesion
in vitro (fresh samples),
supported EC
proliferation (5 days)
41. W.L. Gore &
Associates, Inc.
Carmeda Bioactive Surface
Technology (CBAS�):
layered PEI/dextran
sulfate/glutaraldehyde,
aldehyde activated heparin [44]
Propaten� PTFE
vascular graft
Canine carotid artery
interposition model:
3 months.
Canine model: [
graft patency versus
ePTFE control grafts
and no change in
heparin activity [67].
Yes
42. Randomized clinical
trial: 1 year for bypass
for lower limb ischemia
Clinical trial: Y risk
of primary graft
failure by 37%
compared with PTFE [42].
Jotec GmbH Flowline Bipore� Heparin:
electrostatic bonding
interactions/protein substrate
PTFE vascular graft Clinical trial comparing
femoropoplitea bypass
(2004-present)
NA European CE Mark
Approval only
Maquet Cardiovascular Bioline� coating: recombinant
albumin and covalently
attached heparin
Fusion Bioline�
43. vascular
graft, ePTFE and
PET (Dacron�)
FINEST Phase 3 clinical
trial for peripheral
artery disease
(2011-ongoing)
NA European CE Mark
Approval only
Intervascular Inc.
(Acquired by
Maquet in 2009)
Heparin-bonded
collagen coating
Intergard� composed
of PET (Dacron�)
vascular grafts
Prospective randomized
clinical trial: 5 years for
44. above-knee femoropopliteal
bypass [40].
No difference in
primary patency at
5 years compared
with human umbilical
vein grafts [40].
Yes
Prospective randomized
clinical trial: 5 years for
above/below-knee
femoropopliteal bypass [41].
Significantly improved
patency at 3 years,
but no difference at
5 years compared
with PTFE grafts [41].
Perouse Medical Heparin bioactive
luminal coating
45. PM� Flow Plus Heparin
vascular graft, ePTFE
NA NA European CE Mark
Approval only
NA ¼ not available; PEI ¼ polyethylenimine. Note: Other
clinically available heparin-bonded biomaterials include:
Duraflo�II (Baxter International Inc.), Photolink�
(SurModics Inc.) and Astute� Advanced Heparin Coating
(BioInteractions Ltd.) marketed as Trillium Biosurface�
(Medtronic Inc.) as hemocompatible coating technologies, but
are not currently used for vascular grafts.
R.A. Hoshi et al. / Biomaterials 34 (2013) 30e41 39
Furthermore, heparin is capable of inhibiting platelet adhesion
and activation in the presence of ATIII, but may cause platelet
aggregation under certain conditions depending on the
molecular
weight fraction and concentration [56]. POCeHeparin vascular
grafts described herein significantly inhibited platelet adhesion
as
verified by LDH activity and SEM imaging. Additionally,
hydrophilic
surfaces are associated with improving biocompatibility while
46. inhibiting platelet adhesion and activation [57]. In this regard,
the
POCeHeparin coating dramatically improved the wettability of
the
ePTFE vascular graft surface. The covalently attached heparin
molecule via the diaminohexane linker may create an ideal
hydrophilic layer for further inhibiting platelet adhesion.
Although
POC has been previously shown to inhibit platelet adhesion,
POC
cross-linking and rinsing conditions can affect POC surface
energy
and charge density therefore affecting its interaction with
platelets
when in contact with blood [16]. Therefore, incorporating
heparin
into the POC to provide a more robust inhibition of platelet
adhe-
sion is warranted.
One of the reasons for the poor patency of small-caliber ePTFE
grafts is due to intimal hyperplasia resulting from the migration
and over proliferation of vascular smooth muscle cells [58].
Therefore, the POCeHeparin material was characterized for in
vitro
47. compatibility of HASMCs because of the involvement of
smooth
muscle cell pathology in cardiovascular disease and vascular
graft
failure. POC and POCeHeparin surfaces were capable of
reducing
HASMC proliferation and elevating expression of smooth
muscle
a-actin protein. These results are important because increased
HASMC proliferation and reduction in contractile phenotype
markers such as a-actin, are implicated in stenosis progression
leading to graft failure [59]. Furthermore, heparin signaling and
substrate compliance are known to alter HASMC proliferation
and
phenotype [60,61]. Our findings are noteworthy in that they are
the
first to show that poly(diol citrate) elastomers modified with
heparin are capable of modulating the phenotype of vascular
smooth muscle cells in possible combination with heparin
signaling and polymer substrate compliance and warrant further
investigation regarding the interactions between HASMCs and
elastomeric poly(diol citrate) biomaterials.
Although previous studies have demonstrated good endothelial
cell and BOEC compatibility with unmodified POC surfaces, the
48. covalent modification with heparin and its resulting effects on
cell
behavior must be investigated [13,15]. POCeHeparin surfaces
R.A. Hoshi et al. / Biomaterials 34 (2013) 30e4140
supported adhesion, spreading and proliferation of both BOECs
and
HUVECs. Although the presence of heparin seems to have had
an
effect on HUVEC proliferation, cells were viable and
maintained an
endothelial cell phenotype. Nitric oxide secretion is an
important
endothelial cell mediated process for maintaining a
physiologically
healthy endothelium and inhibiting thrombus formation. Endo-
thelial function was further confirmed by verifying the
production
of NO. BOECs and HUVECs had comparable NO production
when
cultured on POCeHeparin.
There is a limited amount of information in the literature
49. regarding the simultaneous characterization of vascular graft
surfaces enhanced with antithrombogenic activity and the
resulting
influence on endothelialization and smooth muscle cell
function. It
is also well known that novel biomaterials used in vascular
grafts
may also adversely affect endothelial cell function [62].
Moreover,
EPC seeding strategies for improving vascular graft
thromboresist-
ance have heavily relied on the incorporation of collagen, fibrin
and
fibronectin for improving cell compatibility [63e65]. However,
these extracellular matrix and plasma proteins also promote
platelet
adhesion and thrombus formation and a subconfluent or denuded
endothelialized surface may provide nucleation sites for
thrombus
formation. Therefore, the development of POCeHeparin as a
multi-
functional biomaterial is a significant step towards improving
vascular graft performance since it is capable of inhibiting
platelet
adhesion, blood coagulation and vascular smooth muscle cell
50. growth while simultaneously supporting endothelialization.
5. Conclusion
In this report we describe a new approach to impart heparin-
mediated thromboresistance and vascular cell compatibility to
vascular grafts. The POCeHeparin-coated vascular grafts
remained
bioactive and significantly inhibited whole blood clotting and
platelet adhesion. POCeHeparin supported BOEC proliferation
and
expression of endothelial cell-specific phenotype markers and
the
production of nitric oxide. Furthermore, POCeHeparin
modulated
HASMC phenotype via elevated contractile protein expression
and
decreased cell proliferation rate. Our results support the
feasibility
of using BOECs and mature endothelial cell types for ex vivo or
in
situ endothelialization strategies. Due to the ease of synthesis
and
fabrication, the strategy described herein can be readily adopted
to
modify other types of devices such as stents, heart valve
51. replace-
ments devices and hemodialysis tubing.
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GORE-TEX vascular graft performance by Carmeda BioActive
surface heparin
immobilization. Eur J Vasc Endovasc Surg 2003;25(5):432e7.
The blood and vascular cell compatibility of heparin-modified
68. ePTFE vascular grafts1. Introduction2. Materials & methods2.1.
Synthesis and preparation of POC2.2. Heparin immobilization
to POC2.3. Immobilized heparin quantification and contact
angle measurements2.4. Heparin detection by X-ray
photoelectron spectroscopy (XPS)2.5. Isolation of blood
outgrowth endothelial cells from human peripheral blood2.6.
Bioactivity and antithrombogenicity assessment of POC–
Heparin ePTFE grafts2.6.1. Re-calcified whole blood
clotting2.6.2. Platelet adhesion2.7. Effect of immobilized
heparin on vascular cells2.7.1. Cell proliferation2.7.2. Cell
viability2.7.3. Cell phenotype2.7.4. Nitric oxide production2.8.
Statistical analysis3. Results3.1. Heparin immobilization to
POC3.2. Fabrication of the POC–Heparin ePTFE graft3.3.
Bioactivity and antithrombogenicity assessment of POC–
Heparin ePTFE grafts3.3.1. Re-calcified whole blood
clotting3.3.2. Platelet adhesion3.4. Effect of immobilized
heparin on vascular cells3.4.1. HUVEC and BOEC viability and
proliferation on POC–Heparin3.4.2. HASMC viability and
proliferation on POC–Heparin3.4.3. Nitric oxide production4.
Discussion5. ConclusionReferences