Creation of tissue engineering heart valve from human mesenchymal stem cells

Creation of tissue engineering heart valve from human mesenchymal stem cells@程永庆$Dept Thorac Cardiac Surg,Changhai Hosp,2nd Mil Med Univ,Shanghai 200433     

Development and potential of a biomimetic chitosan/type Ⅱ collagen scaffold for cartilage tissue engineering

Background Damaged articular cartilage has very limited capacity for spontaneous healing. Tissue engineering provides a new hope for functional cartilage repair. Creation of an appropriate cell carrier is one of the critical steps for successful tissue engineering. With the supposition that a biomimetic construct might promise to generate better effects, we developed a novel composite scaffold and investigated its potential for cartilage tissue engineering.Methods Chitosan of 88% deacetylation was prepared via a modified base reaction procedure. A freeze-drying process was employed to fabricate a three-dimensional composite scaffold consisting of chitosan and type Ⅱcollagen. The scaffold was treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. Ultrastructure and tensile strength of the matrix were carried out to assess its physico-chemical properties. After subcutaneous implantation in rabbits, its in vivo biocompatibility and degradability of the scaffold were determined. Its capacity to sustain chondrocyte growth and biosynthesis was evaluated through cell-scaffold co-culture in vitro. Results The fabricated composite matrix was porous and sponge-like with interconnected pores measuring from 100－250 μm in diameter. After cross-linking, the scaffold displayed enhanced tensile strength. Subcutaneous implantation results indicated the composite matrix was biocompatible and biodegradable. In intro cell-scaffold culture showed the scaffold sustained chondrocyte proliferation and differentiation, and maintained the spheric chondrocytic phenotype. As indicated by immunohistochemical staining, the chondrocytes synthesized type Ⅱ collagen. Conclusions Chitosan and type Ⅱ collagen can be well blended and developed into a porous 3-D biomimetic matrix. Results of physico-chemical and biological tests suggest the composite matrix satisfies the constraints specified for a tissue-engineered construct and may be used as a chondrocyte carrier for cartilage tissue engineering.     

The mechanism of clinical effectiveness of human fetal liver cell transfusion.

During the development of human fetus, all organs consisting of various types of cells are in rapid growth. The significance of these cells and the chemical components involved, especially those of special functions in medical treatment of certain refractory diseases, have been noticed gradually. The questions as a step further are therefore raised, e.g. what is the mechanism underlying the clinical effectiveness and how to produce the active substances in large amount by biological engineering technique or chemical synthetic method, instead of using the crude preparation directly derived from fetus.     

In Vitro Chondrogenic Phenotype Differentiation of Bone Marrow-derived Mesenchymal Stem Cells

In order to study the chondrogenic phenotype differentiation of adult sheep bone marrowderived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering, MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCS were induced with H-DMEM containing TGF-β3 .IGF-I, Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindlelike appearance to a polynal shape, a large amount of endoplasmic reticulum, Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when induced in vitro and can be used as optimal seed cells for cartilage tissue engineering.     

A Brief Introduction of Shanghai Tissue Engineering Key Laboratory

Shanghai Tissue Engineering Key Laboratory was found in 1997 and is the first tissue engineering research and development center in China, which integrates various areas of basic researches with clinical application of engineered tissues. The center comprises Embryonic Stem Cell Laboratory, In vitro Fertilization Center, Good Manufacture Product (GMP) level human cell culture center, animal cell culture room, Molecular Biology Laboratory, Histology Laboratory, Biomaterial and Bioreactor Laboratory and Biomechanics Laboratory.     

Molecular mechanism of endothelial cells about adhesion and proliferation in tissue engineering heart valve of seeding human umbilical vein endothelial cells on decellular porcine aortic valve

Objective To create the tissue engineering heart valve （TEHV） by seeding human umbilical vein endothelial cells （HUVECs） on decellular porcine aortic valve and investgate its mechanism of endothelial cells in adhesion and proliferation.Methods The porcine aortic valve was acellularized by using trypsin、EDTA、Triton X-100、RNase and DNase treatment.Biomechanical characteristics of fresh valves and acellularized valves were tested.     

Preparation of PLGA Electrospun Nanofibers for Tissue Engineering Applications

Non-woven poly （lactic-co-glycolic acid） （PLGA） nanofibers were prepared by electrospinning technology. The morphology of the electrospun fibers was observed using scanning electron Microscopy （SEM） and the fiber diameter was calculated. Primary human dermal fibroblasts （HDFs） were cultured to evaluate the biocompatibility of the electrospun PLGA nanofibers. The results of MTT testing and SEM showed that the PLGA nanofibers have the morphology of interwoven and highly porous structure and can support the attachment and proliferation of HDFs. It is can be expected to be a potential scaffold material for tissue engineering.     

Tissue Engineering of Heart Valves： Endothelial Cells Differentiated from Endothelial Progenitor Cells Seeding of Acellularized Porcine Valves

Objective Valvular heart disease is an important source of morbidity and mortality world-wide. Classical replacement surgery involves the implantation of mechanical valves or biological valves. However, they all have drawbacks. Tissue engineering of heart valves represents a new experimental approach to improve current modes of therapy in valvular heart surgery. The aim of this study was to develop a new method for a tissue engineered heart valve combining endothelial cells differentiated from endothelial progenitor cells （EPCs）, and a xenogenic acellularized matrix. Methods Porcine aortic valves were acellularized by deterging cell extraction using Triton X-100 and tripsin. Endothelial progenitor cells were isolated from ovine bone marrow by Ficoll gradient centrifugation and cultured in endothelial basal medium containing growth factors. After proliferation and differentiation cells were analyzed by immunohistochemistry and seeded onto the acellular matrix. Analysis of acellularity was performed by light microscopy and scanning electron microscopy. Analysis of the repopulated grafts included immunohistochemistry, histology, and scanning electron microscopy. Results The acellularization procedure resulted in an almost complete removal of the original cells while the 3D matrix was loosened at interfibrillar zones. However the 3D arrangement of the matrix fibers was maintained. Preseeding differentiated endothelial progenitor cells indicated constant endothelial phenotypes including acetylated low density lipoprotein, vascular endothelial growth factor receptor-2, and cluster of differentiation 31 （CD31）, CD34, yon Willebrand factor expression. Seeded endothelial cells differentiated from endothelial progenitor cells established favorable cell-to-matrix attachment, and were positive for cluster of differentiation 31, yon Willebrand factor. Conclusion Porcine aortic valves can be completely acellularized by a detergent-enzymatic extraction procedure. The xenogenic matrix was reseeded with endothelial cells differentiated from endothelial progenitor cells. This approach may eventually lead to the engineering of tissue heart valves repopulated with the patients own autologous cells.     

Isolation, Culture and Identification of Neural Stem Cells in New-born Rats

The cortexes were obtained from new-born rats and dissociated to single cells by triturating. The ceils were cultured in neural stem cell (NSC) culture medium (DMEM supplemented with bFGF, EGF and B27) and formed primary neurospheres after 7 days. Single ceils dissociated from neurosphere were cultured in 96-well plates and formed single-cell cloning neurosphere 7 days later.The primary and single-cell cloning neurospheres were both positive for the immunofluorescent staining of nestin and were identified as NSC. It was proved that NSC can be expanded in vitro and orovide seed cells for neural tissue engineering.     

Osteogenic Potential of Cultured Bone Marrow Stromal Cells Transfected with Transforming Growth Factor β_1 Gene in vitro

Bone tissue engineering has a broad prospect intreating bone defects. It is a crucial link to chooseand optimize seed cells.In recent years,bone mar-row stromal cells(BMSCs) have been regarded to beone kind of preferred seed cells[1,2 ] . We have trans-fected BMSCs with TGF- β1gene and then examinedtheirosteogenic potential in orderto improve theirbi-ological properties.1　 MATERIALSAND METHODS1 .1　 Experimental MaterialsThe materials used in the experiment includedpc DNA3 - T…     

Potential Oxidative Stress in the Bodies of Electric Arc Welding Operators:Effect of Photochemical Smog

To investigate whether photochemical smog emitted during the process of electric arc welding might cause oxidative stress and potential oxidative damage in the bodies of welding operators. Methods Seventy electric arc welding operators (WOs) and 70 healthy volunteers (HVs) were enrolled in a randomized controlled study design, in which the levels of vitamin C (VC) and vitamin E (VE) in plasma as well as the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), and the level of lipoperoxide (LPO) in erythrocytes were determined by spectrophotometry. Results Compared with the average values of the above experimental parameters in the HVs group, the average values of VC and VE in plasma as well as those of SOD, CAT and GPX in erythrocytes in the WOs group were significantly decreased (P<0.005-0.0001), while the average value of LPO in erythrocytes in the WOs group was significantly increased (P<0.0001). The findings from the partial correlation analysis on the controlling of age suggested that with a prolonged duration of exposure to photochemical smog the values of VC, VE, SOD, and GPX, except for CAT, in the WOs were decreased gradually (P<0.05-0.005), the value of LPO in the WOs was increased gradually (P<0.001), and that with the ozone dose increased in the air in each worksite VC, VE, SOD, CAT and GPX decreased (P<0.005-0.001), but LPO increased (P<0.001). The findings from the reliability analysis for the VC, VE, SOD, CAT, GPX, and LPO values which were used to reflect oxidative stress and potential oxidative damage in the WOs showed that the reliability coefficients' alpha (6 items) was 0.8021, P<0.0001, and that the standardized item alpha was 0.9577, P<0.0001. Conclusion Findings in the present study suggest that there exists an oxidative stress induced by long-term exposure to photochemical smog in the bodies of WOs, thereby causing potential oxidative and lipoperoxidative damages in their bodies.     

Ozone Emitted During Copying Process -A Potential Cause of Pathological Oxidative Stress and Potential Oxidative Damage in the Bodies of Operators

INTRODUCTION Copying of references, documents, information and photos plays important roles in social, economic, business, scientific and technological affairs, however, photochemical smog emitted during this process may become risk factors inducing potential oxidative 1Correspondence should be addressed to Prof. Jun-Fu ZHOU, Second Affiliated Hospital of College of Medicine, Zhejiang University, 88 Jiefang R…     

Oxidative stress and free radical damage in patients with acute dipterex poisoning

Objective To investigate whether acute dipterex poisoning (ADP) may cause oxidative stress and free radical damage in the bodies of acute dipterex poisoning patients (ADPPs), and to explore the mechanisms by which ADP may cause oxidative stress and free radical damage. Methods Fifty ADPPs and fifty healthy adult volunteers (HAVs) whose ages, gender and others were matched with the ADPPs were enrolled in a randomized controlled study, in which concentrations of nitric oxide (NO), vitamin C (VC), vitamin E (VE) and P-carotene (P-CAR) in plasma as well as concentration of lipoperoxide (LPO), and activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and acetylcholinesterase (AChE) in erythrocytes were determined by spectrophotometric analytical methods. Results Compared with the average values of experimental parameters in the HAVs group, the average values of plasma NO and erythrocyte LPO in the ADPPs group were significantly increased (P<0.0001), while those of plasma VC,     

Abnormal reactions of free radicals and oxidative damages in the bodies of patients with chronic glomerulonephritis

Objective To study the abnormal reactions of a series of free radicals and the oxidative damages induced by free radical abnormal reactions in the bodies of patients with chronic glomerulonephritis. Methods Eighty chronic glomerulonephritis patients (CGNP) and eighty healthy adult volunteers (HAV) were enrolled in a random control study, in which concentrations of nitric oxide (NO) in plasma, lipoperoxides (LPO) in plasma and in erythrocytes, and vitamin C (VC),vitamin E (VE) and beta-carotene (β-CAR) in plasma as well as activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in erythrocytes were determined with spectrophotometric assays. Results Compared with the average values of the above biochemical parameters in the HAV group, the average values of NO in plasma, and LPO in plasma and erythrocytes in the CGNP group were significantly increased (P = 0.0001), while those of VC, VE and β-CAR in plasma as well as those of SOD, CAT and GPX in erythrocytes in the CGNP group were significantly decreased (P = 0.0001). Pearson product-moment correlation analysis showed that with increase of the concentration of blood creatinine as well as prolongation of the course of disease in the CGNP, the concentrations of NO in plasma, and LPO in plasma and erythrocytes in the CGNP increased gradually, while the concentrations of VC, VE and β-CAR in plasma as well as the activities of SOD, CAT and GPX in erythrocytes in the CGNP decreased gradually (P = 0.002454 - 0.000001).The relative risk ratio (RR) of the above biochemical parameters reflecting oxidative damages in the bodies of CGNP ranged from 6.061 to 72.429. The reliability coefficient (alpha) that the above biochemical parameters were used to reflect the oxidative damages of the CGNP was 0.8137,standardized item alpha = 0.9728, Hotelling's T-Squared = 1135680.191, F = 53274.6478, P =0.000001. Conclusions The findings irt this study show that in the bodies of CGNP a series of free radical chain reactions result in severe pathological aggravation and induce oxidative damages in their bodies. Therefore, suitable dose of antioxidants should be supplemented to them so as to alleviate oxidative damages in their bodies.     

Oxidative Stress before and after Operation in Patients with Chronic Cholecystitis Containing Gallstone

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Oxidative stress before and after operation in patients with chronic cholecystitis containing gallstone

To study the relationship of oxidative, antioxidative constituents and antioxidases in blood with chronic cholecystitis containing gallstone, levels of lipoperoxides (LPO), nitric oxide (NO), vitamin C(VC), vitamin E (VE) and beta-carotene (beta-CAR) in plasma as well as level of LPO, activities of superoxide dismulase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in erythrocytes were investigated by spectrophotometric assay in 107 patients with this condition (PCg) and 100 healthy volunteers (HVs). Compared with HVs group, the average value of LPO and NO in plasma and that of LPO in erythrocytes of PCg group were significantly increased (P < 0.0001), while that of VC, VE and beta-CAR in plasma and the average activities of SOD, CAT and GSH-Px in erythrocytes were significantly decreased (P < 0.0001). Linear regression and correlation analysis for 107 preoperative PCg showed that the value of LPO and NO in plasma and that of LPO in erythrocytes of PCg gradually increased (P < 0.0001), representing a significant linear positive correlation. The value of VC, VE and beta-CAR in plasma and that of SOD, CAT and GSH-Px in erythrocytes of PCg gradually decreased (P < 0.0001), representing a significant linear negative correlation. Stepwise regression and correlation analysis for 107 preoperative PCg suggested that the closest correlation was observed between the course of disease and the value of NO and VC in plasma and that of SOD, GSH-Px and LPO in erythrocytes, r = 0.7306, F = 32.1408, P < 0.0001. Compared with the preoperative PCg group, the average value of LPO and NO in plasma and that of LPO in erythrocytes of the postoperative PCg group were significantly decreased (P < 0.0001). Furthermore, the average value of VC in plasma and that of SOD, CAT and GSH-Px in erythrocytes of the postoperative PCg group were significantly increased (P < 0.0001), whereas no significant difference was found between their average value of VE and beta-CAR in plasma. These findings suggested that oxidative stress was an aggravating pathological condition in PCg group. Therefore, we recommend that in treating PCg, antioxidants such as VC, VE, beta-CAR should be given in order to alleviate their potential oxidative damages.     

3,4—Methylenedioxymethamphetamine（MDMA） Abuse Markedly Inhibits Acetylcholinesterase Activity and Induces Severe Oxidative Damage and Liperoxidative Damage

Objective To investigate whether 3,4-methylenedioxymethamphetamine (MDMA) abuse produces another neurotoxicity which may significantly inhibit the acetylcholinesterase activity and result in severe oxidative damage and liperoxidative damage to MDMA abusers. Methods 120 MDMA abusers (MA) and 120 healthy volunteers (HV) were enrolled in an independent sample control design, in which the levels of lipoperoxide (LPO) in plasma and erythrocytes as well as the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and acetylcholinesterase (AChE) in erythrocytes were determined by spectrophotometric methods. Results Compared with the average values of biochemical parameters in the HV group, those of LPO in plasma and erythrocytes in the MA group were significantly increased (P<0.0001), while those of SOD, CAT, GPX and AChE in erythrocytes in the MA group were significantly decreased (P<0.0001). The Pearson product-moment correlation analysis between the values of AChE and bioche     

Increased oxidative stress and damage in patients with chronic bacterial prostatitis

Objective To investigate whether chronic bacterial prostatitis （CBP） increases oxidative stress and damage in patients with CBP, and to explore its possible mechanism. Methods Eighty patients with CBP and 80 healthy adults as controls were enrolled in a case-control study, in which levels of nitric oxide （NO）, vitamin C （VC）, and vitamin E （VE） in plasma, as well as malondialdehyde （MDA）, activities of superoxide dismutase （SOD）, and eatalase （CAT） in erythrocytes were determined by spectrophotometry. Results Compared with the average values of NO, VC, VE, MDA, SOD, and CAT in the healthy control group, those of plasma N O and erythrocyte MDA in the CBP group were significantly increased （P〈0.00 1）, and those of plasma VC and VE as well as erythrocyte SOD and CAT in the CBP group were significantly decreased （P〈0.001）. Findings from partial correlation analysis for course of the disease and NO, VC, VE, MDA, SOD, and CAT in 80 patients with CBP, adjusted for age, suggested that with prolonged course of the disease, values of NO and MDA were gradually increased （P〈0.001）, and those of VC, VE, SOD, and CAT were gradually decreased （P〈0.05-0.001）. The findings from stepwise regression analysis for course of the disease and NO, VC, VE, MDA, SOD, and CAT in CBP group suggested that the model of stepwise regression was Y = -19.1160 ＋0.3112MDA ＋ 0.0337NO, F = 22.1734, P〈0.001, r = 0.6045, P〈0.001. The findings from the reliability analysis for VC, VE, SOD, CAT, NO, and MDA in the CBP group showed that the reliability coefficients＇ alpha （6 items） was 0.7195, P〈0.0001, and the standardized item alpha was 0.9307, P〈0.0001. Conclusion There exist increased oxidative stress and damage induced by chronic bacterial prostatitis in patients, and such a phenomenon is closely related to the course of disease.     

Increased oxidative stress in women with pregnancy-induced hypertension

Objective To investigate whether pregnancy-induced hypertension （PIH） may increase oxidative stress in women with PIH, and to explore the mechanisms by which PIH may increase oxidative stress and potential free radical damage. Methods Seventy women with PIH and seventy women with uncomplicated normotensive pregnancy （UNP） whose age, nutritional conditions, levels of hemoglobin and albumin were all matched, were enrolled in a randomized controlled trial. Their plasma concentrations of nitric oxide （NO）, vitamin C （VC）, vitamin E （VE）, and β-carotene （β-CAR） as well as their erythrocyte malondialdehyde （MDA）, and activities of superoxide dismutase （SOD）, catalase （CAT） and glutathione peroxidase （GPX） were determined by spcctrophotometry. Results Compared with average values of the above experimental parameters in the women with UNP, the average value of erythrocyte MDA in the women with PIH significantly increased （P〈0.0001）, while the average values of plasma NO, VC, VE, and β-CAR as well as those of erythrocyte SOD, CAT, and GPX in the women with PIH significantly decreased （P〈0.0005-0.0001）. The findings from partial correlation analysis （controlling for age） for 70 women with PIH showed that with elevated systolic blood pressure （SBP） and diastolic blood pressure （DBP）, MDA value gradually increased （P〈0.001）, and NO, VC, VE, β-CAR, SOD, CAT, and GPX values gradually decreased （P〈0.02-0.001）. The findings from reliability analysis for NO, VC, VE, β-CAR, SOD, CAT, GPX, and MDA values used to reflect increased oxidative stress and potential free radical damage in women with PIH showed that the reliability coefficients （alpha, 8 items） = 0.7062, P〈 0.0001, and the standardized item alpha = 0.9116, P〈 0.0001. Conclusion The findings in the present research suggest that pregnancy-induced hypertension can increase oxidative stress and potential free radical damage in women with pregnancy-induced hypertension.     

May Chronic Childhood Constipation Cause Oxidative Stress and Potential Free Radical Damage to Children？

INTRODUCTION Constipation is a symptom rather than a specific disease. It is generally defined by patients as defecation frequency of twice weekly or less, and the defecation frequency of patients with chronic constipation is much less still. However, the…