Compatibility in vitro of albumin-heme (O(2) carrier) with blood cell components

Recombinant human serum albumin including 2-[8-[N-(2-methylimidazolyl)]octanoyloxymethyl]-5,10,15,20-tetrakis(alpha,alpha,alpha,alpha-o-pivaloylamino)phenylporphinatoiron(II) (albumin-heme; rHSA-FeP) is a synthetic hemoprotein that has sufficient capability to reversibly bind and release O(2) under physiological conditions (pH 7.3, 37 degrees C) similar to hemoglobin and myoglobin. In order to use this albumin-based O(2) carrier as a new class of red blood cell substitutes, its compatibility with blood cell components carefully was investigated in vitro. After the addition of the rHSA-FeP solution into whole blood at 10, 20, and 44 vol %, the FeP concentration in the plasma phase remained constant for 6 h at 37 degrees C in each group, and no significant time dependence was observed in the numbers of red blood cells, white blood cells, or platelets. The microscopic observations clearly showed that the shapes of the red blood cells had not been deformed during the measurement period. With respect to the blood coagulation parameters (prothrombin time and activated partial thromboplastin time), the coexistence of rHSA-FeP had only a negligibly small influence. Also the blood compatibility under dynamic flow conditions was evaluated using a microchannel array flow analyzer. All these results suggest that the albumin-heme has no effect on the morphology of blood cell components in vitro.     

煅烧骨的安全性

背景：经高温处理的煅烧骨具有类似自然骨的连续微孔结构,良好的生物相容性和降解性。 目的：观察牛煅烧骨的生物相容性、细胞相容性及毒性。 方法：①细胞相容性实验：将牛煅烧骨与第3 代已诱导的Wistar大鼠骨髓间充质干细胞复合培养。②溶血实验：将煅烧骨浸提液、生理盐水与双蒸水加入兔血中。③凝血实验：将煅烧骨加入兔血浆中。④急性毒性实验：在昆明种小鼠尾静脉分别注射煅烧骨浸提液、生理盐水。⑤微核实验：在小鼠腹腔分别注射煅烧骨浸提液、生理盐水与环磷酰胺。⑥局部刺激性实验：将煅烧骨浸提液、生理盐水分别注射于兔两侧脊柱皮下。⑦热源检测实验：在兔耳静脉注射煅烧骨浸提液。⑧皮下植入实验：将煅烧骨材料植入Wistar大鼠背部皮下。 结果与结论：煅烧骨材料无细胞毒性,具有良好的细胞及血液相容性;对皮肤、肌肉无刺激作用;对心、肝、肾重要器官无毒性作用;皮下植入后对周围组织无刺激作用,能够部分降解吸收并被机体组织替代;无致热作用,对凝血功能无影响,对小鼠骨髓细胞无抑制及毒性作用。     

Effect of solute-coupled volume absorption on oxygen consumption in cat ileum.

The effects of solute-coupled volume absorption on blood flow, oxygen consumption, and vascular resistance were analyzed in autoperfused segments of cat ileum. Intestinal absorption was stimulated by placing either Tyrode solution, Tyrode + glucose, or Tyrode + taurocholate into the ileal lumen. Net volume absorption rates (Jv,m) were determined using a volume recovery method. Oxygen consumption (VO2) increased during the absorption of all solutions. The absorption of Tyrode solution plus glucose caused the greatest increase in VO2, whereas Tyrode plus taurocholate resulted in the smallest increase. For Tyrode solution and Tyrode plus glucose absorption, the increased VO2 was due predominantly to an increased blood flow, whereas the increased VO2 with taurocholate resulted from an increased oxygen extraction. A linear relationship between the change in VO2 during transport and Jv,m was aquired for Tyrode solution, and Tyrode + glucose. The results indicate that the oxygen requirements of the absorbing intestine are dependent on both the rate of transport and the solutes being transported.     

Precipitation of serum proteins by polyethylene glycol (PEG) in pretransfusion testing

Polyethylene glycol (PEG) is used as a potentiator of blood group antigen-antibody interactions. Although PEG is known to precipitate immunoglobulins, we could find no reports of this reagent entrapping red blood cells (RBCs) in irreversible clumps. The patient we describe here had hyperglobulinemia with a reversed albumin:globulin ratio and a diffuse immunoglobulin peak on serum protein electrophoresis. During preparation of serologic tests, a precipitate formed that entrapped the RBCs when PEG was added. Rapid recognition of this phenomenon could prevent delay in the selection of blood for transfusion by substituting PEG-indirect antiglobulin test (IAT) with another technique such as low-ionic-strength solution (LISS)-IAT, and by increasing the number of washes prior to addition of the antiglobulin reagent.     

Effects of heparin immobilization on the surface characteristics of a biological tissue fixed with a naturally occurring crosslinking agent (genipin): an in vitro study

Heparinized biomaterials have been used to manufacture blood-contacting prostheses. The present study was intended to characterize the surface properties of a genipin-fixed biological tissue immobilized with heparin using the methods of ionic binding (the /h-i tissue) or covalent binding via multi-point attachment (the /h-m tissue) or end-point attachment (the /h-e tissue). The surface characteristics of test tissues evaluated were water contact angle, surface tension, protein adsorption, platelet adhesion, and cellular compatibility. Nonheparinized and the glutaraldehyde-fixed counterparts were used as controls. It was found that immobilization of heparin on the glutaraldehyde- and genipin-fixed tissues increased their hydrophilicity and surface tension and suppressed their mole ratio of adsorbed fibrinogen to adsorbed albumin and the amount of platelets adhered. Among the heparinized tissues, the /h-m tissue was more hydrophobic and had a higher mole ratio of adsorbed fibrinogen to adsorbed albumin and a greater amount of platelets adhered than the /h-i and /h-e tissues. In general, the surface characteristics of the /h-i tissue were comparable to the /h-e tissue. However, it is known that the ionically immobilized heparin may be displaced from the surface by an ion-exchange mechanism when exposed to blood. There were no significant differences in hydrophilicity, surface tension, the mole ratio of adsorbed fibrinogen to adsorbed albumin, and the amount of platelet adhesion between the glutaraldehyde- and genipin-fixed tissues in comparison with their respective counterparts. However, the cellular compatibility of the genipin-fixed tissues with or without heparinization was significantly superior to its glutaraldehyde-fixed counterparts.     

Inhibition of platelet adhesion to glow discharge modified surfaces

Plasma glow technique has created much interest in the field of surface modification of polymers due to its versatility of generating active polar groups on the surface without affecting the bulk properties. Here an attempt is made to inter-relate the surface properties and platelet adhesion on various polymeric substrates due to plasma treatments. Initially, a critical review of the process and development of thrombosis upon contact of an artificial surface with blood, has been provided, which has been extended with the need for surface modifications to improve their blood compatibility and the versatility of plasma treatments for such modifications have been emphasized. Phospholipids like phosphoryl choline, phosphatidyl choline and phosphoryl ethanolamine were attached to Angioflex surface by plasma glow. The role of such modified substrates to interact with platelets were investigated using Tyrode washed calf platelets. It seems, glow discharge modified phosphoryl choline bilayers dramatically inhibited the platelet-surface binding, which may be due to their biochemical resemblance with thromboresistant surfaces of human blood cells. Further, the behaviour of all phospholipids towards bloodpolymer interaction is not similar and may change depending on the nature of their functional groups, net charge of the phospholipid adsorbed surface and their interaction with platelets and its activation. It is possible to chemically immobilize lipid bilayers on standard polymers, using plasma glow, to improve their biological performance; by suitably selecting the phospholipid combinations.     

New vessel formation after surgical brain injury in the rat's cerebral cortex II. Formation of the blood vessels distal to the surgical injury

We investigated the features of newly formed blood vessels after surgical brain injury of the rat's cerebral cortex distal to the operated region. We document the process of split mature blood vessels by an endothelial bridge and morphological features of newly formed vessels. We did not observe a disruption of brain parenchyma. The endothelial lining in vessels was complete. The morphological features of the endothelial cells and basement membrane show that non-sprouting angiogenesis takes place distally to the surgical injury.     

The effect of Tris-buffered media and Tyrode physiologic saline solution on the antigenic release of histamine from human leukocytes.

Allergic histamine release from leukocytes was compared in three different media: Tyrode physiologic saline solution, Tris-buffered saline containing human albumin, calcium, and magnesium (Tris-ACM), and Tris-ACM with homologous serum. In a selected group of low histamine releasers, the maximal amount of antigenic histamine release was significantly higher in Tyrode solution as compared to Tris-ACM buffer. When homologous serum was added to Tris-acm, an enhancement of histamine release greater than with Tyrode solution was obtained. These results suggest that Tris-ACM may not be the optimal buffer for leukocyte histamine release experiments. Since Tyrode solution contains no serum proteins that bind slow-reacting substance of anaphylaxis or prostaglandins, the use of this medium may be advantageous for the study of the release of the chemical mediators from human leukocytes.     

Initiation of vagal control of heart rate in the embryonic chick

The objective was to determine on what day of development the vagal complex i.e., dorsal motor nucleus in the floor of the fourth ventricle, vagus nerve and post synaptic neuron assumes functional control of the heart rate in the chick embryo. Adapting a technique used by Armstrong ('35) for the Fundulus embryo, 0.003 ml of Tyrode solution containing 0.03 μg of acetylcholine was injected into the fourth ventricles of 16 chick embryos at four days of age, 25 at five and 15 at six days of age. Heart rates were taken before and after injection. Analyzed statistically no significant changes in heart rate occurred following injection of acetylcholine into four day embryos but at five days a significant average drop from 106 to 95 beats per minute occurred. The same was true in six-day-old embryos where the average drop in rate was from 108 before to 84 after injection. As proof that the acetylcholine injected into the brain ventricle acted by stimulating the vagal mechanism and not by diffusing into cerebral blood vessels and being carried to the heart we found that corresponding amounts of acetylcholine injected into omphalomesenteric veins had no significant effect on heart rate. Also 0.003 ml of plain Tyrode solution injected into the fourth ventricle elicited no significant response. These observations justify the conclusion that the vagal pathway to the heart becomes functional between five and six days of development in the chick embryo.     

医用Ta5+掺杂TiO2生物薄膜材料的合成与性能研究

利用射频控溅射技术合成Ta^5 掺杂的TiO2薄膜材料。采用X射线衍射（XRD）和电子能谱（XPS）等技术对薄膜的成分和结构进行了分析,并利用动态凝血时间测定法和血小板粘附试验研究了薄膜的血液相容性,同时对薄膜的硬度、耐磨性等力学特性进行了研究和评价。结果表明,Ta^5 掺杂的TiO2薄膜不仅具有良好的血液相容性,同时还具有较优的力学耐久性能。     

A perfused canine right bundle branch-septal model for electrophysiological studies.

As documented in the literature, isolated ventricular muscle preparations perfused with protein-free media become edematous and deteriorate. We hypothesized that the addition of colloid oncotic pressure to the perfusion medium might affect favorably the electrical characteristics and survival of an isolated right bundle branch-septal muscle preparation perfused via the anterior septal artery. Perfusion with protein-free Tyrode solution produced edema formation and a loss of transmembrane potential of Purkinje and muscle cells within 5.9 +/- 0.40 h. Addition of bovine albumin, 4 g/100 ml, to the Tyrode solution or perfusion with horse serum greatly reduced the formation of edema. Furthermore, intact electrical activity continued for more than 9 +/- 0.31 h (P less than 0.001). The preparations showed a greater diastolic potential and action potential amplitude during perfusion than during superfusion with Tyrode solution. In conclusion: 1) a viable, perfused septal muscle preparation was developed for electrophysiological studies, and 2) colloid oncotic pressure in the perfusion medium is essential to preserve the integrity of the capillary circulation and to eliminate edema formation and tissue death.     

Bioactivity and Platelet Adhesion Study of a Human Thrombomodulin-Immobilized Nitinol Surface

Nitinol is a newly developed biomaterial that is gaining popularity in many biomedical applications. It has been reported that nitinol would not induce an inflammatory response and repulsion by the immunization after implantation in the human body. Besides, nitinol is a kind of shape memory alloy, which can memorize shapes at different temperatures. This can improve the convenience in surgery. However, nitinol has poor blood compatibility, so that further modification was needed to improve the antithrombogenicity. Human thrombomodulin (hTM), an endothelial-cell-associated glycoprotein, can be considered as a natural potent anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. In this study, the surface of nitinol was pre-activated by utilizing silanization with amino-terminated silane. The incorporated amino groups were available for the subsequent covalent immobilization of hTM by 2,4,6-trichloro-1,3,5-triazine (TCT), the coupling reagent. The surface density of immobilized hTM was determined by the Bradford method. The bioactivity of immobilized hTM and blood compatibility of various nitinol substrates were evaluated by the protein C activation assay and platelet adhesion test. It was observed that the immobilized hTM still had the ability to enhance protein C activation, though its activity was lower than the free hTM in solution. Furthermore, the platelet adhesion test showed that only a few platelets were adhered on the hTM-immobilized nitinol substrate. Therefore, the immobilization of thrombomodulin onto nitinol substrate could improve the blood compatibility of nitinol and might have the potential of application in antithrombogenic medical applications.     

Albumin marks pseudopodia of astrocytoma cells responding to hepatocyte growth factor or serum

It is well accepted that dysfunction in the blood brain barrier (BBB) allows permeation of albumin from the bloodstream into astrocytic brain tumors, especially glioblastomas, the most aggressive astrocytomas. In vitro, bovine serum albumin (BSA) aids functional cell assays by maintaining cytokines and growth factors in solution and delivering its cargo of fatty acids. Earlier, we showed that BSA was prominent in lysates prepared from pseudopodia formed by U87 astrocytoma cells. The present studies investigated the association of albumin with pseudopodia formed by U87 and LN229 astrocytoma cells. With hepatocyte growth factor (HGF) stimulation, cell migration was enhanced and BSA, especially its dimerized form, was prominent in pseudopodia compared to unmigrated cells on one-dimensional gels and immunoblots. When lysates were equalized for levels of glyceraldehyde-3-phosphate dehydrogenase, the rise for BSA levels in pseudopodia vs migrated cells was comparable or greater than levels noted for established pseudopodial proteins, beta-actin and ezrin. The increase for dimerized BSA in pseudopodia compared to unmigrated cells was greater than the rise in levels of beta-actin, ezrin, HGF, and phosphorylated Met when pseudopodia were harvested from filters with 1 mum pores using either cell line. Fluorescein (F)-labeled BSA co-localized with HGF on actin-rich cellular protrusions and with CM-DiI labeled pseudopodial plasma membranes. The F-BSA highlighted small, individual pseudopodial profiles more so than complex pseudopodial networks (reticulopodia) or unmigrated cells. Labeled human serum albumin also decorated pseudopodia preferentially. Albumin's association with pseudopodia may help to explain its selective accumulation in astrocytomas in vivo. The leaky BBB permits serum albumin to enter the microenvironment of astrocytomas thus allowing their invasive cells contact with serum albumin as a source of fatty acids that would be useful for remodeling cell membranes in pseudopodia. Thus, albumin potentially aids and marks invasion as it accumulates in these tumors.     

热解碳人工心脏瓣膜材料表面改性研究

对氮离子注入热解碳及在热解碳表面离子束增强沉积钛氧化物薄膜进行了研究,采用Ｘ光电子能谱仪,俄歇电子能谱仪测定了表面改性层的成价和价态,用四探针方法测定了表面电阻率,用接触角方法测定了材料表面能,用动态凝血时间及血小板粘附评价了表面改性层的血液相容性。研究表明,热解碳经表面Ｎ离子注入后有碳氧化合物形成,其血液相容性有所改善,而采用离子束增强沉积合成ＴｉＯ２－ｘ薄膜则使热解碳备注相 容性获得了显著改善     

A simple technique for isolating healthy heart cells from mouse models

Single heart cells of mouse models provide powerful tools for heart research. However, their isolation is not easy, and it imposes a significant bottleneck on their use in cellular studies of the heart. Aiming to overcome this problem, this report introduces a novel technique that reproducibly isolates healthy heart cells from mouse models. Using simple devices that ensure easy handling and the rapid aortic cannulation of a small mouse heart, cell isolation was done under physiological conditions without using the "KB" medium or 2,3-butanedione monoxime (BDM). The isolated cells consistently had a healthy appearance and a high viability of 75 +/- 5% (mean +/- SD) in Tyrode solution containing 1.8 mM Ca2+. After 8 h of storage at 37 degrees C, they still had a viability of 45 +/- 12%. The cells showed normal contraction properties when field-stimulated, and they generated normal action potentials and membrane currents under the whole-cell clamp condition. The beta-adrenergic signal transduction of the cells was also normal when it was examined with the isoproterenol enhancement of the L-type Ca2+ current.     

Evaluations of blood compatibility via protein adsorption treatment of the vascular scaffold surfaces fabricated with polylactide and surface-modified expanded polytetrafluoroethylene for tissue engineering applications

Blood compatibility was evaluated by short-term in vitro blood perfusion on candidate vascular scaffold surfaces of a biodegradable, porous polylactide scaffold and a chemically surface-modified expanded polytetrafluoroethylene (ePTFE) over a clinical ePTFE, by measuring blood cell adhesion either directly or after adsorption treatment with albumin and fibrinogen. The results indicated that the extent of blood cell adhesion was affected by scaffold surface properties and pre-adsorption of proteins such as fibrinogen and albumin. Surface morphologies and porosity of the scaffolds were characterized by scanning electron microscopy and porosimetry, and the amount of fibrinogen and albumin adsorbed on the scaffolds was measured and verified by employing radiolabeled C(14) albumin and I(125) fibrinogen by a scintillation counter and a gamma counter, respectively. Even though treatment of fibrinogen adsorption on the samples in advance led to higher induction of blood cell adhesion than those with no fibrinogen adsorption, the polylactide scaffold surface itself induced highest amount of the adhered blood cells in this study judged by analyses of their surface area. These results would be employed as guidance in determining a choice of the implant methods, in vitro versus in vivo tissue engineering, of the novel chemically modified ePTFE and the biodegradable polylactide scaffolds.     

Drug-Carrying Albumin Film for Blood-Contacting Biomaterials

Surface-induced thrombosis is a major complication in the development of blood-contacting medical devices. Serum albumin has the ability to bind to a wide variety of compounds, including drugs, and neither cells nor proteins adsorb to an albumin-coated surface. These properties of albumin are useful for improving the blood compatibility of biomaterial surfaces. In the present study, we prepared a water-insoluble film by cross-linking pharmaceutical grade recombinant human serum albumin aiming to the clinical applications, and loaded the film with a synthetic antiplatelet drug, cilostazol. The resultant film possessed native albumin characteristics such as drug binding ability and resistance to cell adhesion. Mouse fibroblast L929 cells did not adhere on the albumin film, just as they did not adhere on native albumin-coated surfaces. Furthermore, when the albumin film carrying cilostazol was placed in PBS containing Tween-80, the release of cilostazol was sustained over 144 h. The results indicate that the surface coating with thus prepared albumin film can confer the biomaterials with antithrombogenic surface by virtue of its non-adhesiveness to cells and its release of cilostazol.     

Effects of veratrine on repolarization in the canine right bundle branch.

Superfusion of Purkinje fibers in the canine right bundle branch with Tyrode solution containing veratrine, less than or equal to 1 mug/ml, prolonged repolarization phases 2 and 3 and induced a long-lasting negative after-potential that ended in some experiments with a slightly hyperpolarized resting transmembrane potential. The alterations in repolarization time were reversed by washout with veratrine-free Tyrode solution or by adding tetrodotoxin to the veratrine-containing Tyrode solution to a concentration of 0.1 and 1 mug/ml. The effect of veratrine on the repolarization time was more prominent at slower driving rates. Its effect on repolarization of ventricular muscle cells was smaller than that on the repolarization of Purkinje fibers. In the concentrations used, veratrine did not induce multiple responses.     

聚醚砜中空纤维膜血浆分离器血浆分离功能与血液相容性的评价

对新型材料聚醚砜制作的中空纤维膜血浆分离器进行动物实验 ,评价了膜对血浆蛋白的分离功能及材料的血液相容性。分离过程中 ,实验动物状况良好 ,无溶血现象发生 ,膜对血浆总蛋白、白蛋白和球蛋白的筛分系数均在 95以上 ,约 6 0的血浆从全血中分离出来。白细胞、血小板和四种凝血因子在分离开始时都有不同程度地减少 ,但均在临床允许的范围内。     

Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification

Endothelial cell seeding constitutes an appreciated method to improve blood compatibility of small-diameter vascular grafts. In this study, we report the development of a simple innovative technique based on multilayered polyelectrolyte films as cell adhesive substrates. Polyelectrolyte multilayered films ending by poly(sodium-4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) or poly(L-glutamic acid)/poly(D-lysine) (PGA/PDL) could enhance cell adhesion by modification of the physico-chemical properties of the surface. The biological responses of human umbilical vein endothelial cells seeded on the polyelectrolyte multilayer films, on PDL or PAH monolayers, and on control surfaces, were evaluated in terms of initial attachment, growth, cellular metabolic activity, endothelial phenotype, and adhesion. The results showed that polyelectrolyte multilayers neither induce cytotoxic effects nor alter the phenotype of the endothelial cells. The polyelectrolyte multilayered films enhanced initial cell attachment as compared to the polyelectrolyte monolayer. Cell growth observed on the films was similar to that on TCPS. Among the different coating tested, the film ending by PSS/PAH exhibited an excellent cellular biocompatibility and appeared to be the most interesting surface in terms of cellular adhesion and growth. Such films could be used to cover hydrophobic (cell resistant) substrates in order to promote cell colonization, thereby constituting an excellent material for endothelial cell seeding.