Acute and chronic canine ex vivo blood interactions with NHLBI-DTB primary reference materials

Thrombus deposition was measured on NHLBI-DTB Primary Reference Material polyethylene (PRM-PE) and polydimethylsiloxane (PRM-SR) and their commercially available counterparts, surgical grade Intramedic polyethylene and Dow Corning Silastic. Canine blood-contacting experiments evaluating short-term (up to 60 min) and longer-term (up to 24 h) thrombus deposition were used to quantitate adherent platelets on the lumenal surface of test materials ex vivo. A similar pattern of thrombus deposition and detachment was observed for all materials in both acute and chronic blood contact. Although differences in the wall shear rates affected the absolute numbers of adherent platelets, the relative levels of thrombus deposition showed similarities between the two experiments, with the polyethylene materials as a group showing slightly less deposition than the silicone rubber materials. The PRM-PE showed the least thrombus deposition at extended exposure to blood. The PRM-SR showed the most thrombus deposition in the acute term. The overall similarity in blood compatibility and surface properties indicates the need for the inclusion of less thromboresistant and more polar reference materials.     

Effect of surface hydrophilicity on ex vivo blood compatibility of segmented polyurethanes

The relationship between surface, bulk and ex vivo blood-contacting properties of segmented polyurethanes with various polyol soft segment was investigated. The polyols used in this study were poly(ethylene oxide), poly(tetramethylene oxide), hydrogenated poly(butadiene), poly(butadiene) and poly(dimethylsiloxane). The hard segment of these segmented polyurethanes was composed of 4,4' diphenylmethane diisocyanate and 1,4 butanediol, present at 50 wt%. An experimental polyurethane, Biostable PUR, which has shown excellent biostability, was used in this study. The segmented polyurethanes based on the hydrophobic polyols such as poly(dimethylsiloxane) and hydrogenated poly(butadiene) showed distinct microphase separation between hard and soft segments. X-ray photoelectron spectroscopy revealed the surface enrichment of the hydrophobic component at the air-solid interface. Dynamic contact angle measurements indicated that the poly(dimethylsiloxane)-based segmented polyurethane possessed a hydrophobic surface in water. The poly(dimethylsiloxane)-based segmented polyurethane had the lowest platelet adhesion among the segmented polyurethanes investigated in this study, whilst the platelet deposition on the poly(ethylene oxide)-based polymer increased with time.     

In vitro and ex vivo blood compatibility study of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer-coated hemodialysis hollow fibers

To identify the advantages of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer-coated polysulfone (PSf) hollow fibers for hemodialyzer and hemofilter minimodules with hollow fibers were made and blood compatibility was evaluated in vitro and ex vivo. Three types of hollow fibers, i.e., pure PSf (no additives), PSf alloyed with poly(1-vinyl-2-pyrrolidone) (PVPy), and PSf coated with the MPC copolymer, were processed in wet conditions. Commercially available hollow fibers (APS) were used as a control sample. The PSf hollow fibers have a condensed structure. A porous structure was observed when the PVPy was alloyed before wet processing, and no effect of the innercoated MPC copolymer on the porous structure was observed. One-tenth-sized minimodules of the conventional hemodialyzer were fabricated with 200 fibers each. The solute permeability of the hollow fibers was evaluated using 10% bovine serum in a buffer solution containing cytochrome C, which is a model protein of ₂-microglobulin. After circulation for 2.5h, the solute permeability of APS and PVPy-alloyed PSf hollow fibers decreased to 50% compared with their initial values. In contrast, the value for the hollow fibers innercoated with the MPC copolymer maintained its initial level. The inner surface of the dialysis membranes was observed with a transmission electron microscope and a layer of adsorbed protein on the PSf, APS, and PVPy-alloyed PSf hollow fibers was observed, but not on the MPC copolymer-coated fibers. Blood cell adhesion was then evaluated by circulation of whole rabbit blood without any anticoagulant ex vivo. Many adherent cells were observed on the PVPy-alloyed PSf hollow fibers; however, blood cells did not adhere or aggregate on the MPC copolymer-coated hollow fibers. From these results, we concluded that the in-situ coating of MPC copolymer on PSf hollow fibers is effective in preventing blood coagulation and maintaining the solute permeability of the fibers.     

人工血管材料生物相容性和血液相容性的研究与分析☆

背景：随着生物医学工程学和生物材料学的发展,人工血管的研究得到广泛的应用,人工血管材料得到不断更新,如何提高血管的通畅性和人工血管材料的相容性是在近年来人工血管研究的重点。 目的：利用CNKI数据库文献检索和深度分析功能,对于人工血管材料学研究的文献资料趋势进行多层次探讨分析。 设计：文献计量学分析。 资料提取：以电子检索方式对CNKI数据库2002-01/2011-12有关人工血管材料学研究的文献进行分析,采用检索词为“人工血管(Artificial blood vessels);生物材料(Biomaterials)”,对检索的相关文献运用数据库中自带的分析功能和Excel软件绘制图表的功能进行分析,通过文字和图表的形式将统计和计量数据分析,描述其分布特征。 入选标准：纳入标准：①与人工血管材料相关的基础研究论文。②与人工血管材料临床应用相关的论文。排除标准：①与文章目的无关的文献。②重复研究的文献。③刊社信息。④未发表的文章。⑤需电话追踪和手工检索逐一分析的文章。⑥年鉴。 主要数据判定指标：以CNKI数据库学术期刊文献出版时间、文献数量、学科类别、研究机构、来源期刊、文献被引频次、文献下载频次、关联文献、作者分布、基金资助情况和主要关键词进行相关分析。并对CNKI数据库中博士学位论文、优秀硕士学位论文、专利技术和重要会议论文进行分析。 结果：在CNKI数据库学术期刊收录2002/2011的文献中,共检索到122篇与人工血管材料学研究相关的文献。文献数量产出趋于上升趋势,2008年文献产出数量最多共17篇;《中国组织工程研究与临床康复》杂志发表文献量最多为27篇,占全部文献的22.1%;人工血管材料学研究基金资助以国家自然科学基金项目为主,共发表文献30篇;人工血管材料学研究以聚氨酯的研究为主,突出材料学生物相容性和血液相容性的研究。 结论：通过文献计量学方法对CNKI数据库学术期刊关于人工血管材料的文献进行分析,可为中国从事人工血管材料学基础研究和临床实施的医务工作者进一步确定科研思路提供有价值的参考。     

In vitro, ex vivo, in vivo veritas

In vitro assessments of inhaler performance are important in product development and quality control, but are not, as such, good predictors of performance in vivo . It is, however, possible to modify in vitro techniques so that they more closely resemble the in vivo situation. Measurements of fine-particle dose (defined as the amount of drug with an aerodynamic diameter less than 5 μm) by cascade impactor have shown that the measured fine-particle dose in vitro is highly dependent on the geometry of the inlet to the impactor, the fine-particle dose being considerably lower when the cast of a human throat (an "anatomical throat") is used than when a standard glass inlet is used. This difference is, however, less with a dry-powder inhaler such as Turbuhaler® than with a pressurized metered-dose inhaler (pMDI). Furthermore, there is a good correlation between fine-particle dose measured in vitro and in vivo lung deposition, provided that an anatomically correct inlet is used for the in vitro determination. Studies in children have shown that the degree of lung deposition of budesonide, delivered via Turbuhaler, is of the same order of size as the in vitro fine-particle dose. No comparable data are available for pMDIs; however, since children are smaller than adults, it is likely that differences in lung deposition between Turbuhaler and pMDIs are probably greater in children than in adults.     

A model of human whole blood lymphokine release for in vitro and ex vivo use

Endotoxin (lipopolysaccharide, LPS) inducible cytokine release by human whole blood is increasingly used to model inflammatory responses in vitro, to detect the presence of pyrogenic contaminations as well as to monitor disease states or immunomodulatory treatments ex vivo. However, the LPS-stimulated blood model primarily allows the assessment of monocyte responses. Here, a whole blood model was established which allows assessment of lymphocyte responses. Four different superantigens, namely staphylococcal enterotoxin A and B (SEA, SEB), toxic shock syndrome toxin-1 (TSST-1) or streptococcal exotoxin A (SPEA) were tested with respect to the induction of lymphokine release. All superantigens were capable of inducing significant amounts of the lymphokines interferon-gamma (IFNgamma), interleukin 2 (IL-2), IL-4, IL-5, IL-13 and tumor necrosis factor beta (TNFbeta) after 72 h of incubation. Concentration-dependencies and kinetics were determined. Blood from 160 healthy donors was used to assess the variability of SEB-inducible lymphokine release. Interindividual differences were more pronounced compared to LPS-inducible monokine release. However, the individual response was maintained when blood from six donors was tested once a week for 8 weeks, suggesting that the individual response represents a donor characteristic. The model appears to be suitable for the evaluation of immunomodulatory agents in vitro as well as ex vivo.     

In vivo leucocyte interactions with the NHLBI-DTB primary reference materials: Polyethylene and silica-free polydimethylsiloxane

In vivo leucocyte interactions with the NHLBI-DTB primary reference materials, low density polyethylene (LDPE) and silica-free polydimethylsiloxane (PDMS), were qualitatively and quantitatively characterized using a cage implant system over a 21 d implantation period. Scanning electron microscopy (SEM) and cytochemical staining procedures were utilized to observe the cellular events occurring at the leucocyte/ biomaterial interface. The results showed that more cells adhered to the PDMS surface than the LDPE surface at days 4 and 7. The differential analysis revealed that mononuclear cells, presumably macrophages, preferentially adhered to both polymer surfaces. By day 21, there were more very large (> 20 nuclei per cell) foreign body giant cells (FBGCs) present on the PDMS surface than the LDPE surface. The phagocytic capabilities of the adhered cells, including the FBGCs, decreased to a greater extent on the PDMS surface, corresponding to the earlier and more extensive spreading of these cells observed in the morphological analysis.     

Evaluation of the in vivo and ex vivo optical properties in a mouse ear model

Determination of in vivo optical properties is a challenging problem. Absorption and scattering measured ex vivo are often used for in vivo applications. To investigate the validity of this approach, we have obtained and compared the optical properties of mouse ears in vivo and ex vivo in the spectral range from 370 to 1650 nm. Integrating sphere spectrophotometry in combination with the inverse Monte Carlo technique was employed to determine absorption coefficients, mu(a), scattering coefficients, mu(s), and anisotropy factors, g. Two groups of mice were used for the study. The first group was measured in vivo and ex vivo within 5-10 min post mortem. The second group was measured in vivo and ex vivo every 24 h for up to 72 h after sacrifice. Between the measurements the tissues were kept at 4 degrees C wrapped in a gauze moistened with saline solution. Then the specimens were frozen at -25 degrees C for 40 min, thawed and measured again. The results indicate that the absorption coefficients determined in vivo and ex vivo within 5-10 min post mortem differed considerably only in the spectral range dominated by hemoglobin. These changes can be attributed to rapid deoxygenation of tissue and blood post mortem. Absorption coefficients determined ex vivo up to 72 h post mortem decreased gradually with time in the spectral regions dominated by hemoglobin and water, which can be explained by the continuing loss of blood. Absorption properties of the frozen-thawed ex vivo tissues showed increase in oxygenation, which is likely caused by the release of hemoglobin from hemolyzed erythrocytes. Scattering of the ex vivo tissues decreased gradually with time in the entire spectral range due to the continuing loss of blood and partial cell damage. Anisotropy factors did not change considerably.     

In vitro and ex vivo effect of tiaprofenic acid on human peripheral blood mononuclear cells.

The effect of the non-steroidal anti-inflammatory drug (NSAID) tiaprofenic acid on different human immune parameters was investigated in vitro or following in vivo administration in healthy adult volunteers. Results from the in vitro study demonstrated an increased mitogen-induced blastogenesis and interleukin 2 (IL-2) production together with a reduced polyclonal immunoglobulin (Ig) secretion in the presence of the drug. Results from the ex vivo study showed that treatment with tiaprofenic acid had no significant effects on the immune parameters investigated, i.e. unstimulated and mitogen-induced proliferation and IL-2 production, spontaneous and stimulated Ig synthesis, lymphocyte subpopulations, serum Ig and complement levels.     

Blood compatibility of polymers:in vitro andin vivo tests

Synthetic materials used for medical prostheses and devices, mainly those which will come into contact with blood, have to provide for specific properties. Three in vitro blood-compatibility tests are presented; one is a screening method (m.t.e.g.) whose results show remarkable correlations to in vivo behaviour, the other two (Nosé-blood-chamber, protein adsorption) yield more basis information on synthetic surfaces. Aspects of toxicity and biodegradation should be included in the definition of ‘blood compatibility’. After preselection by in vitro tests, a material has to pass an in vivo examination in animal experiments (shunt, catheter, tube, test chamber) under haemodynamic conditions. Finally, devices or prostheses in their intended shape, such as the total artificial heart, are implanted in animals.     

Relevance of ex vivo blood lymphocyte assay for in vivo lymphocyte function

Determinations of in vitro proliferative and secretory activities of peripheral blood cells are used widely for research in clinical immunology but, to our knowledge, have not been evaluated as to their power to reflect in vivo activities quantitatively. Here, we addressed this question by quantitatively correlating the in vitro secretion of interleukin (IL)-5 by peripheral blood cells to the in vivo activity of IL-5 as reflected by peripheral-blood eosinophil counts. Studying 458 humans exposed to transmission of the nematode Onchocerca volvulus, IL-5 was measured in the supernatants of 0.02-ml whole-blood cells cultured in the presence of O. volvulus extract or mitogen. O. volvulus-reactive IL-5 secretion was correlated significantly to blood eosinophilia in a quantitative manner explaining 15.1% (95% CI 8.3-19.9%) of the variability of eosinophil counts. Interestingly, correlations were obtained only if parasite counts were included in the calculation using multiple regression analysis. The results show that in vitro assays of minute amounts of blood lymphocytes may quantitatively reflect activities of the entire lymphocyte population in vivo.     

Transplantation of ex vivo expanded cord blood.

Umbilical cord blood (CB) from unrelated donors is increasingly used to restore hematopoiesis after myeloablative therapy. CB transplants are associated with higher rates of delayed and failed engraftment than are bone marrow transplants, particularly for adult patients. We studied the ex vivo expansion of CB in an attempt to improve time to engraftment and reduce the graft failure rate in the recipients. In this feasibility study, 37 patients (25 adults, 12 children) with hematologic malignancies (n = 34) or breast cancer (n = 3) received high-dose therapy followed by unrelated allogeneic CB transplantation. A fraction of each patient's CB allograft was CD34-selected and cultured ex vivo for 10 days prior to transplantation in defined media with stem cell factor, granulocyte colony-stimulating factor, and megakaryocyte growth and differentiation factor. The remainder of the CB graft was infused without further manipulation. Two sequential cohorts of patients were accrued to the study. The first cohort had 40% and the second cohort had 60% of their CB graft expanded. Patients received a median of 0.99 x 10(7) total nucleated cells (expanded plus unexpanded) per kilogram. The median time to engraftment of neutrophils was 28 days (range, 15-49 days) and of platelets was 106 days (range, 38-345 days). All evaluable patients who were followed for 28 days or longer achieved engraftment of neutrophils. Grade III/IV acute GVHD was documented in 40% and extensive chronic GVHD in 63% of patients. At a median follow-up of 30 months, 13 (35%) of 37 of patients survived. This study demonstrates that the CD34 selection and ex vivo expansion of CB prior to transplantation of CB is feasible. Additional accrual will be required to assess the clinical efficacy of expanded CB progenitors.     

Comparison of ex vivo and in vitro human fibroblast ageing models

Several studies have analyzed modulation of gene expression during physiological ageing with interesting, but often contradictory results, depending on the model used. In the present report we compare age-related metabolic and synthetic parameters in human dermal fibroblasts (HDF) isolated from young and old subjects (ex vivo ageing model) and cultured from early up to late cumulative population doublings (CPD) (in vitro ageing model) in order to distinguish changes induced in vivo by the aged environment and maintained in vitro, from those associated with cell senescence and progressive CPD. Results demonstrate that fibroblasts from aged donors, already at early CPD, exhibit an impaired redox balance, highlighting the importance of this parameter during ageing, even in the presence of standard environmental conditions, which are considered optimal for cell growth. By contrast, several proteins, as those related to heat shock response, or involved in endoplasmic reticulum and membrane trafficking, appeared differentially expressed only during in vitro ageing, suggesting that, at high CPD, the whole cell machinery becomes permanently altered. Finally, given the importance of the elastic component for a long-lasting connective tissue structural and functional compliance, this study focuses also on elastin and fibulin-5 synthesis and deposition, demonstrating a close relationship between fibulin-5 and ageing.     

In vitro blood compatibility of glycosaminoglycan-precipitated collagens.

Precipitation of bovine hide collagen by chondroitin 6-sulfate at low pH and subsequent crosslinking enhances the blood compatibility of native collagen. Both dehydrothermal crosslinking and complexation with chrondroitin 6-sulfate separately decrease the platelet-aggregating activity of collagen. Crosslinking also decreases the number of free acidic and free basic residues on collagen, which suggests that crosslinking involves these residues in condensation reactions with formation of intrachain and interchain synthetic peptide bonds. Clotting times for collagen precipitated with chondroitin 6-sulfate indicate that this surface does not activate or interfere with coagulation via either the intrinsic or extrinsic pathway. These findings support further consideration of collagen modified by chondroitin 6-sulfate as a blood compatible material.     

A tyrosine-rich amelogenin peptide promotes neovasculogenesis in vitro and ex vivo

The formation of new blood vessels has been shown to be fundamental in the repair of many damaged tissues, and we have recently shown that the adult human periodontal ligament contains multipotent stem/progenitor cells that are capable of undergoing vasculogenic and angiogenic differentiation in vitro and ex vivo. Enamel matrix protein (EMP) is a heterogeneous mixture of mainly amelogenin-derived proteins produced during tooth development and has been reported to be sometimes effective in stimulating these processes, including in clinical regeneration of the periodontal ligament. However, the identity of the specific bioactive component of EMP remains unclear. In the present study we show that, while the high-molecular-weight Fraction A of enamel matrix derivative (a heat-treated form of EMP) is unable to stimulate the vasculogenic differentiation of human periodontal ligament cells (HPC) in vitro, the low-molecular-weight Fraction C significantly up-regulates the expression of the endothelial markers VEGFR2, Tie-1, Tie-2, VE-cadherin and vWF and markedly increases the internalization of low-density lipoprotein. Furthermore, we also demonstrate, for the first time, that the synthetic homolog of the 45-amino acid tyrosine-rich amelogenin peptide (TRAP) present in Fraction C is likely to be responsible for its vasculogenesis-inducing activity. Moreover, the chemically synthesized TRAP peptide is also shown here to be capable of up-regulating the angiogenic differentiation of the HPC, based on its marked stimulation of in vitro cell migration and tubule formation and of blood vessel formation assay in a chick embryo chorioallantoic membrane model ex vivo. This novel peptide, and modified derivatives, might thereby represent a new class of regenerative drug that has the ability to elicit new blood vessel formation and promote wound healing in vivo.     

Lysophosphatidic acid enhances antimycobacterial activity both in vitro and ex vivo

Lysophosphatidic acid (LPA) is a polar lipid metabolite which is involved in a wide range of biological processes, including cell proliferation and migration, wound healing, and increase of endothelial permeability. The present study reports evidences showing that LPA is able to enhance the antimicrobial activity of human macrophages and of bronchoalveolar lavage cells from tuberculosis patients leading to intracellular growth control of Mycobacterium tuberculosis. Such antimicrobial activity is mediated by the activation of phospholipase D which in turn induces acidification of M. tuberculosis containing phagosomes and is associated with the enhanced expression of Cathepsin D. These results suggest the possible protective role of this lysophospholipid in the activation of innate antimycobacterial response.     

血液相容性抗凝血生物医用高分子材料的制备及其机制

背景:由于生物医用材料要接触人体内环境,甚至必须植入生物体内,因此要求具有无毒性、优良的生物相容性、高化学稳定性、合适的物理机械性能以及易加工成型性。 目的：从生物惰性材料、生物活性表面和白蛋白的结构及其在抗凝血上的应用几个方面分析血液相容性抗凝血生物医用高分子材料的制备及其机制。 方法：由第一作者检索1969/2010 PubMed数据及万方数据库有关血液相容性抗凝血生物医用高分子材料的制备及其机制等方面的文献。 结果与结论：目前抗凝血材料的制备基本上只是采用单独的生物惰性表面或生物活性表面,虽然都获得了较好效果,但不能长期保持其生物相容性尤其是血液相容性,如果能将惰性表面与活性表面结合起来,使材料同时具备两者的长处,并能充分利用人体血液中的天然组分白蛋白或许会是抗凝血材料的一个发展趋势。今后希望通过采用高生物惰性的PEU和具有生物活性的白蛋白识别因子cibacron blue复合,合成具有优良性质的活性改性物,并以此对聚氨酯进行改性。     

A comparison of ex vivo cytokine production in venous and capillary blood

We performed a randomized study of the immunological effects of an early measles vaccine given at 4.5 months of age and aimed to obtain venous samples from the infants at baseline and 6 weeks later. If this was not feasible, a capillary sample was obtained. We analysed baseline samples from the first 50 children enrolled in the study to investigate the potential differences in ex vivo cytokine production between venous blood and capillary blood. We also obtained paired venous and capillary blood samples from 11 adult volunteers. Whole blood was stimulated with lipopolysaccharide (LPS) [a Toll-like receptor (TLR)-4 ligand], (S)-(2, 3-bis (palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH, trihydrochloride (PAM3Cys) (a TLR-2 ligand), phytohaemagglutinin (PHA) or purified protein derivative (PPD). Cytokine concentrations in the supernatants were assessed by a multiplexed assay and were compared between venous and capillary samples in both infants and adults. The production of both the pro- and the anti-inflammatory cytokines, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10, was higher in cultures of capillary blood compared with venous blood. This was found in non-stimulated control samples as well as in blood stimulated with PAM3Cys and PPD. Adults produced more IL-5 in venous blood than in capillary blood upon PHA stimulation. We found no other difference in the levels of IL-5 or IFN-gamma between venous and capillary blood. In capillary blood we found sex differences in response to PHA but this was not the case in venous blood. We found significant differences in the production of cytokines between venous and capillary blood. Such differences should be taken into account when setting up immuno-epidemiological studies.     

Histamine receptors on leukocytes are expressed differently in vitro and ex vivo

The effect of histamine and histamine antagonists on the respiratory burst activity of leukocytes was studied. The activity was measured as zymosan-induced luminol-dependent chemiluminescence (CL) of isolated leukocytes and 1:2,500 diluted whole blood (WB). Histamine caused a dose-dependent inhibition of CL. For separated leukocytes the ID50 was 8 x 10(-5) M and for WB it was 1.5 x 10(-3) M. Diphenhydramine, an H1-antagonist increased the inhibitory effect of histamine while H2-antagonist cimetidine blocked the inhibition of CL of separated leukocytes. Cimetidine was not capable of reversing the effect of histamine on WB-CL. These data suggest that the histamine receptors on leukocytes are associated with Fc and/or complement receptors and the expression and function of histamine receptors can be studied by measuring the respiratory burst activity. The results also provide evidence of differences in histamine action in vitro and ex vivo conditions in WB.