体外循环前后心肌和血清地高辛浓度的变化及影响其变化的多因素分析

目的：探讨体外循环对成人心肌和血清地高辛浓度的影响及影响其改变的多因素分析。方法：成年病入术前四四按体重计算的维持剂量的地高辛一周以上,用Park的方法提取心肌内的地高辛,然后再用放免法测定转机前后心肌地高辛含量和血清地高辛浓度;对可能影响其浓度的各因素进行多元回归分析。结果：血清地高辛浓度停机时最低（0．603±．316μg/L）,转机前最高（0．995±0．409μg/L）,停机后4h有回升现象（0．837±0．383μg/L）;心肌地高辛的含量停机时（344M．839±8．935μg/kg）校转机前（25．391±6．538μg/kg）高;血清地高辛浓度和心肌内地高辛含量相关性差（P＞0.05)。多因素分析提示：影响心肌和血清地高辛各浓度的因素各不相同。结论：体外循环使血清地高辛浓度下降,但有回升现象,而心肌中浓度则不下降;地高辛的血清浓度与心肌含量相关性差;影响心肌和血清地高辛浓度改变的因素各不相同。     

Quantitative and qualitative evaluation of blood salvaged after extracorporeal circulation (ECC) in paediatric heart surgery. Study of biochemical, morphological and structural variations of RBC after ECC and after salvaging of ECC circuit priming blood

The salvaging of ECC circuit priming blood is essential for reducing the morbidity related to homologous blood transfusions and the importance of this technique is inversely proportionate to the age and weight of the child. In infants, the washing and centrifugation of blood not only drastically reduce the risk of contracting blood-transmitted diseases and cut management costs, but are also of considerable hemodynamic importance, producing a rapid normalization of the patient's hematocrit and hemoglobin and balancing the O2 consumption/demand ratio. The marketing of miniaturized salvagin devices with 55 ml bowls by Dideco has made possible the recovery of small quantities of blood, so as to normalise the hematic crisis and permit the application of total hemodilution in low-weight patients. The salvaged blood shows an average hematocrit of 52.7+/-9.7% (max 68.1%) and an average hemoglobin of 17.6 +/- 2.9 g/dl (max 20.7 g/dl), and maintains its structural components, osmotic resistance, concentration of intraerythrocytic hemoglobin and mean corpuscular hemoglobin all intact. Washing with isoosmotic and isoionic hydroelectrolytic solutions normalizes the ionic situation in the post-operative period and activated blood salvaging after Extracorporeal Circulation. The use of solutions without nutritional substances results however in a considerable fall in the number of enzymes in the intraerythrocytic metabolic glucide chain (G6PDH: -40.7 +/- 14.3% p<0.001), (PK: -23.8 +/- 20.5% p<0.03). This drop may be responsible for erythrocytic morphological alterations (echinocytic change) and probably for the release of hemoglobin from the red blood cells. Washing with isoionic, isoosmotic solutions containing G5% and adenine could, at least in theory, improve the quality of the salvaged blood, by normalizing the morphology and the volume of the RBC and by increasing the hematocrit.     

Inhibition of bacterial adhesion on PVC endotracheal tubes by RF-oxygen glow discharge, sodium hydroxide and silver nitrate treatments

Medical-grade poly(vinyl chloride) (PVC) was chemically modified to study how the incorporation of monovalent silver influences Pseudomonas aeruginosa adhesion and colonization. The modification investigated consisted of a radio frequency-oxygen (RF-O(2)) glow discharge pre-functionalization, followed by a two-step wet-treatment in sodium hydroxide and silver nitrate solutions. X-ray photoelectron spectroscopy (XPS) analysis and contact angle measurements were used to investigate the chemical nature and surface wettability of the films following each step of the modification. XPS analysis proved that the RF-O(2) plasma pre-functionalization of native PVC reproducibly increased the amount of functional groups representative of PVC additives, including ether/alcohol, esters and carboxyl groups. More specifically, we demonstrated that the O-C=O groups representative of the phthalic ester and zinc carboxylate additives identified for native PVC increased by two-fold following the RF-O(2) plasma pre-functionalization step. Although RF-O(2) pre-functionalization did not have an effect on the silver content of the NaOH/AgNO(3) treated substrates, such a modification was necessary for biomaterial products that did not have reproducible surfaces amongst production lots. XPS analysis also demonstrated that saponification with sodium hydroxide (NaOH) of esters, like those of the phthalic ester additives of PVC is a simple, irreversible method of hydrolysis, which produced sodium carboxylate and sodium phthalate salts. Exposure of native PVC to NaOH resulted in an increased surface hydrophilicity (from ca 90 degrees to ca 60 degrees ) due to dechlorination. XPS analysis following further incubation in silver nitrate demonstrated that silver ions can be trapped when the sodium of sodium carboxylate is replaced by silver after performing a second treatment with a monovalent silver-containing solution. The creation of silver salt on native PVC resulted in an ultra-hydrophobic (>120 degrees ) surface. The chemical modifications using NaOH and AgNO(3) wet treatments completely inhibited bacterial adhesion of four strains of P. aeruginosa to both native and oxygen-pre-functionalized PVC, and efficiently prevented colonization over longer periods (72 h). Our results suggest that surface modifications that incorporate silver ions would be extremely effective at reducing bacterial colonization to medical devices.     

Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition.

The aim of this study was to determine the influence of two key scaffold design parameters, void fraction (VF) and pore size, on the attachment, growth, and extracellular matrix deposition by several cell types. Disc-shaped, porous, poly(-lactic acid) (L-PLA) scaffolds were manufactured by the TheriForm solid free-form fabrication process to generate scaffolds with two VF (75% and 90%) and four pore size distributions (< 38, 38-63, 63-106, and 106-150 microm). Microcomputed tomography analysis revealed that the average pore size was generally larger than the NaCl used, while VF was at or near the designated percentage. The response of three cell types-canine dermal fibroblasts (DmFb), vascular smooth muscle cells (VSMC), or microvascular epithelial cells (MVEC)-to variations in architecture during a 4-week culture period were assessed using histology, metabolic activity, and extracellular matrix deposition as comparative metrics. DmFb, VSMC, and MVEC showed uniform seeding on scaffolds with 90% VF for each pore size, in contrast to the corresponding 75% VF scaffolds. DmFb showed the least selectivity for pore sizes. VSMC displayed equivalent cell proliferation and matrix deposition for the three largest pore sizes. MVEC formed disconnected webs of tissue with sparse extracellular matrix at 90% VF and >38 to 150 microm; however, when cultured on scaffolds with pores formed with salt particles of <38 microm, MVEC formed a multilayered lining on the scaffolds surface. Culture data from scaffolds with a 75% VF suggests that the structural features were unsuitable for tissue formation. Hence, there were limits of acceptable scaffold architecture (VF, pore size) that modulated in vitro cellular responses.     

Poly(ethylene glycol) hydrogel system supports preadipocyte viability, adhesion, and proliferation

The ultimate goal of this research is to develop an injectable cell-scaffold system capable of permitting adipogenesis to abrogate soft tissue deficiencies resulting from trauma, tumor resection, and congenital abnormalities. The present work compares the efficacy of photopolymerizable poly(ethylene glycol) and specific derivatives as a scaffold for preadipocyte (adipocyte precursor cell) viability, adhesion, and proliferation. Four variations of a poly(ethylene glycol) scaffold are prepared and examined. The first scaffold consists of poly(ethylene glycol) diacrylate, which is not susceptible to hydrolysis or enzymatic degradation. Preadipocyte death is observed over 1 week in this hydrogel configuration. Adhesion sites, specifically the laminin-binding peptide sequence YIGSR, were incorporated into the second scaffold to promote cellular adhesion as a prerequisite for preadipocyte proliferation. Preadipocytes remain viable in this scaffold system, but do not proliferate in this nondegradable hydrogel. The third scaffold system studied consists of poly(ethylene glycol) modified with the peptide sequence LGPA to permit polymer degradation by cell-secreted collagenase. No adhesion peptide is incorporated into this scaffold system. Cellular proliferation is initially observed, followed by cell death. The previous three scaffold configurations do not permit preadipocyte adhesion and proliferation. In contrast, the fourth system studied, poly(ethylene glycol) modified to incorporate both LGPA and YIGSR, permits preadipocyte adherence and proliferation subsequent to polymer degradation. Our results indicate that a scaffold system containing specific degradation sites and cell adhesion ligands permits cells to adhere and proliferate, thus providing a potential cell-scaffold system for adipogenesis.     

Presence of plasticizer di-2(ethylhexyl)phthalate in primed extracorporeal circulation circuits

Many centers advocate the use of a standby wet-primed extracorporeal membrane oxygenation (ECMO) circuit for rapid deployment during cardiopulmonary resuscitation. However, concerns with regard to the potential health hazards associated with the release of the plasticizer di-2(ethylhexyl)phthalate (DEHP) from the polyvinyl chloride (PVC) tubing exist. The purpose of this study was to determine the time course of DEHP release from a preprimed ECMO circuit and to evaluate the effect of PVC tubing coatings on DEHP release. Seven circuits including three uncoated (Medtronic, Medtronic with albumin, and Medtronic Super Tygon) and four attenuated surfaces (Carmeda, COBE Smart, Medtronic Trillium, and Terumo x-coated) were primed with Plasmalyte A. Samples of the circuit prime were collected over a period of 2 weeks and were analyzed for DEHP, using gas chromatography. Results were compared by using a two-tailed t test. One coated (Carmeda) and all three uncoated circuits leached DEHP. The greatest amount of leaching occurred in the uncoated Medtronic tubing with albumin. The COBE Smart, Medtronic Trillium, and Terumo x-coated circuits had undetectable amounts of DEHP (p = 0.006 vs Medtronic uncoated). Prepriming an ECMO circuit composed of uncoated PVC tubing is associated with DEHP release. Using coated PVC tubing appears to eliminate DEHP release over a 2-week period.     

RGD peptide-conjugated poly(dimethylsiloxane) promotes adhesion, proliferation, and collagen secretion of human fibroblasts

A novel technique for conjugating Arg-Gly-Asp (RGD) peptides to poly(dimethylsiloxane) (PDMS) surfaces as well as its application to cell culture is presented in this paper. This technique performs RGD conjugation to PDMS through photochemical immobilization of functional NHS groups to PDMS surface followed with linking RGD peptide to the surface via coupling reaction with NHS. A bifunctional photolinker, N-sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), was used to conjugate RGD peptide to the surface. Compared to existing methods for peptide conjugation to PDMS, this technique is convenient, efficient, and free of organic contamination to PDMS surfaces. It can also be used to conjugate other peptides or proteins to most polymeric materials. In addition, cell culture studies showed that the RGD-conjugated PDMS surfaces promoted the adhesion, proliferation, and collagen production of human skin fibroblasts (HSFs). Finally, the RGD-conjugated PDMS surfaces are resistant to autoclaving and UV irradiation, which enables them to be repeatedly used in cell culture studies.     

马来酸酐改性聚乳酸对成骨细胞黏附、增殖和分化的研究

通过研究马来酸酐改性聚乳酸(MPLA)和聚乳酸(PDLLA)材料表面对MC3T3-E1成骨细胞形态、黏附、增殖、细胞总蛋白含量、碱性磷酸酶活性及细胞分泌无机钙含量的影响,评价MPLA和PDLLA材料的细胞相容性。结果显示:与PDLLA相比,MPLA材料上的成骨细胞完全黏附和充分铺展;MPLA材料上细胞的增殖速率、细胞总蛋白含量、细胞碱性磷酸酶活性及细胞分泌的无机钙含量都显著高于PDLLA(P<0.01)。这些结果说明,MPLA材料能促进MC3T3-E1成骨细胞的黏附、铺展、增殖及蛋白质的合成,并能促进成骨细胞的分化和矿化,与PDLLA材料相比具有更好的细胞相容性。     

Contact profilometry and correspondence analysis to correlate surface properties and cell adhesion in vitro of uncoated and coated Ti and Ti6Al4V disks

A fundamental goal in the field of implantology is the design of specific devices able to induce a controlled and rapid "osseointegration". This result has been achieved by means of surface modifications aimed at optimizing implant-to-bone contact; furthermore, bone cell adhesion on implant surface has been directly improved by the application of biomolecules that stimulate new tissue formation, thus controlling interactions between biological environment and implanted materials. Actually, methods for biochemical factor delivery at the interface between implant surface and biological tissues are under investigation; a reliable technique is represented by the inclusion of biologically active molecules into biocompatible and biodegradable materials used for coating implant surface. This paper focuses the application of three polymeric materials already acknowledged in the clinical practice, i.e. poly-L-lactic acid (PLLA), poly-DL-lactic acid (PDLA), and sodium alginate hydrogel. They have been used to coat Ti (Ti2) and Ti6Al4V (Ti5) disks; their characteristics have been determined and their performances compared, with specific regard to the ability in allowing osteoblast adhesion in vitro. Moreover, profilometry data analysis permitted to identify a specific roughness parameter (peak density) which mainly controls the amount of osteoblast adhesion.     

Erythrocyte morphology in anemia after artificial circulation (based on scanning electron microscopic data)

Erythrocyte stereo-ultrastructure of 58 patients operated under the conditions of extracorporeal circulation was studied by scanning electron microscopy. It is established that after the operation with extracorporeal circulation the hemoglobin level is always decreasing, 3 mg% on the average; hemoglobin decrease up to 10 mg% and less was considered as anemia, this occurring according to the approximate calculations, in 10-11% of cases. The causes of anemia in an early postoperative period are as follows: mechanical trauma of erythrocytes in the system of extracorporeal circulation; disturbance of the osmotic balance; alteration of the erythrocyte membrane plasticity due to the change of its enzyme-lipid component. Anemia in the late postoperative period is due to the purulent inflammatory processes. Morphological substrate of anemia is represented by the iron-deficient, target-like, two-pit erythrocytes, spherocytes, lacerated erythrocytes and other forms.     

同型半胱氨酸对血管内皮细胞增殖、贴壁和迁移的影响

目的：探讨同型半胱氨酸（homocysteine，Hcy)对血管内皮细胞生长、增殖、贴壁和迁移的影响。方法:将人脐静脉内皮细胞（HUVEC，6-8代）采用含20%胎牛血清（FBS）、60 mg/ L 内皮细胞生长添加剂（endothelial cell growth supplement，ECGS)、0.005 U/ L 肝素的M199培养。在加入不同浓度的Hcy处理后，用胰酶消化细胞，白细胞计数板计数细胞；同时通过［3H］胸腺嘧啶核苷掺入实验观察Hcy对HUVEC细胞增殖的影响。此外，在加入不同浓度的Hcy处理后，用胰酶消化细胞，重新接种于纤连蛋白包被过的培养皿中，静置培养30 min，洗掉未贴壁细胞，对贴壁细胞进行固定、结晶紫染色、脱色、比色，观察Hcy对细胞贴壁的影响。并进一步通过划伤实验观察Hcy对内皮迁移的影响。结果:100 mmol/L 及以上浓度的Hcy能抑制HUVEC增殖、DNA合成和细胞迁移，200 mmol/L及以上浓度的Hcy能抑制HUVEC贴壁，并且随着剂量的加大，抑制作用都逐渐增强。结论:Hcy对HUVEC细胞增殖、DNA合成、细胞迁移及贴壁均有着不同程度的抑制作用。这可能是Hcy参与动脉粥样硬化发病的机制之一。     

Effect of surface chemistry on bacterial adhesion, viability, and morphology

Helicobacter pylori (H. pylori) is one of the most common infectious agents in the world and it is thought to colonize the gastric mucosa of about half of the world's population causing several gastric diseases. In this work, the effect of surface chemistry on H. pylori nonspecific adhesion, viability, and morphology was evaluated using three H. pylori strains with different adhesins expression profile. Self-assembled monolayers (SAMs) of alkanethiols on gold were used to obtain surfaces exposing different functional groups: OH, CH3, and ethylene glycol (EG4). Bacterial adhesion onto the surfaces reached a plateau at 2 h. There was a correlation between adhesion and the exposed surface group, with bacterial cells adhering preferentially to CH3-SAMs while EG4-SAMs prevented H. pylori adhesion during the entire adhesion test (24 h). Surfaces that presented the EG4 group were also the only ones that significantly reduced the viability of adhered bacteria. Surface chemistry also influenced the morphology of adhered bacteria. The H. pylori rod shape observed in the control (Tissue Culture Polyethylene-TCPE) was only retrieved on CH3-SAMs. This work demonstrates that surface chemistry, namely specific functional groups on the material, influence the nonspecific adsorption of H. pylori. Moreover, the features of the bacterial strain and the surface chemistry can alter the adhesion kinetics, as well as the morphology and viability of attached bacteria.     

血管平滑肌细胞对联合培养内皮祖细胞黏附、增殖与形态的影响

目的：探讨血管平滑肌细胞对联合培养的内皮祖细胞黏附、增殖与分化的影响。方法：从人脐血中分离纯化内皮祖细胞并鉴定；建立内皮祖细胞和平滑肌细胞的联合培养模型；细胞粘附实验测定内皮祖细胞的粘附能力；形态学观察内皮祖细胞的分化与增殖。结果：与血管平滑肌细胞联合培养的内皮祖细胞的粘附能力比单独培养时提高了63％，长梭形细胞数量增加，同时克隆形成单位的数量下降了约60％。结论：血管平滑肌细胞促进了内皮祖细胞的粘附与分化，同时抑制了内皮祖细胞的增殖能力。     

Immunolocalization of the cellular adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule (PECAM), in human edometrium throughout the menstrual cycle

In the present study the presence and distribution of cellularadhesion molecules involved in leukocyte binding were investigatedin human endometrium. Endometrial biopsies (n = 45) were collectedfrom women at all stages of normal menstrual cycles. Consecutivecryostat sections of endometrium were immunostained with monoclonalantibodies to intercellular adhesion molecule-1 (ICAM-1) andplatelet endothelial cell adhesion molecule (PECAM) and haematoxylinand eosin. Primary antibody binding was visualized using a streptavidin–biotinsystem. Strong staining for PECAM was observed in endothelialcells of all vessel types and in focal areas of stroma includingsingle cells, small clusters and larger aggregates of cells.At menstruation, however, almost the entire stroma stained forPECAM which was temporally related to a massive influx of leukocytes.ICAM-1 staining, which was consistently less intense than PECAMstaining, was detected in vascular endothelial cells duringthe cycle, reaching a peak at menstruation. Unlike PECAM, ICAM-1staining did not occur consistently across all vessel types.Stromal staining for ICAM-1 was rare except at menstruation,when almost the entire stroma showed positive staining for ICAM-1.No glandular or luminal epithelial staining was detected foreither PECAM or ICAM-1. This study demonstrates that PECAM andICAM-1 are expressed on endothelial cells of veins, arteriolesand capillaries, and stromal cells within human endometrium.     

Fibroblast adhesion and proliferation on poly(ethylene glycol) hydrogels crosslinked by hydrolyzable polyrotaxane

Fibroblast culture was performed to evaluate cell adhesion and proliferation on poly(ethylene glycol) (PEG) hydrogels crosslinked by a hydrolyzable polyrotaxane. The polyrotaxane consisting of alpha-cyclodextrins (alpha-CDs) and PEG terminated by benzyloxycarbonyl (Z)-L-phenylalanine (L-Phe) via ester linkage was used as a multi-functional crosslinker in the PEG hydrogels. From the results of contact angle and small angle light scattering measurements, it was suggested that the surface and bulk structure of the PEG hydrogels were heterogeneous. Fibroblast adhesion and proliferation on the hydrogels was observed. The number of fibroblast adhesion on the hydrogels crosslinked by the polyrotaxane was proportional to contact angle values and correlation length, and was significantly higher than those crosslinked by alpha-CDs in spite of similar contact angle and correlation length. These findings suggest that the cells recognize the surface heterogeneity due to the polyrotaxane structure, and the number of cell adhesion and proliferation is controllable by the polyrotaxane content in feed.     

Cell morphology, markers, spreading, and proliferation on orthopaedic biomaterials. An innovative cellular model for the "in vitro" study

The aims of tissue engineering are the in vitro reconstruction of functionally active tissues, and the in vivo induction of their appropriate development. The great progresses in the fields of biology and biomaterials represent key events, which allowed the recent improvement of tissue engineering. In the orthopaedic perspective, tissue engineering is focused on the development of innovative materials, whose action consists in recruiting bone progenitor cells and in stimulating their proliferation. In this context, it should remind that these materials should not only allow cells adhesion and proliferation, but also ensure that attached cells maintain the cellular properties of the original tissue. In this study, a new cellular model, suitable for the rapid in vitro determination of the above parameters, is presented. The cell model derives from a human osteosarcoma cell line, Saos-2, which maintained the cytological features of the osteoblast cells. The cell line was genetically modified to express constitutively the enhanced green fluorescent protein. The engineered cell line Saos-eGFP represents a suitable in vitro mode for studying the biocompatibility, the cell adhesion, spreading, and proliferation on biomaterials developed for clinical applications.     

Thrombogenicity is not reduced when heparin and phospholipid bonded circuits are used in a rabbit model of extracorporeal circulation

In an effort to better mimic the thromboresistive nature of vascular endothelium, extracorporeal circuits bonded with heparin or phospholipids were developed. Using no systemic heparinization, these circuits were compared with standard poly(vinyl)chloride (PVC) (Tygon) in a rabbit model of extracorporeal circulation (ECC). Control circuits were run with and without systemic heparinization and used as comparison groups against the test circuits. Two New Zealand White rabbits were used per study: One was used as the platelet donor for 111Indium platelet labeling; the other animal was placed on bicaval ECC for 4 hours. Circuits (heparin coated n = 6, phospholipid coated n = 8, nonheparinized controls n = 14, heparinized controls n = 18) consisted of 1 m of tubing, two downsizing connectors, and two venous cannulae. ECC blood flow was at least 75 ml/min. Platelet and fibrinogen measurements were made hourly, and circuit dosimetry was performed at the end of the study or on circuit thrombosis. Thrombosis of the circuit occurred in one heparin coated, two phospholipid coated, and eight nonheparinized control circuits. None of the heparinized control circuits thrombosed. There was no significant difference between the groups with regard to platelet count or platelet adhesion. Test circuits exhibited preservation of fibrinogen levels. In this rabbit model of ECC, circuits coated with heparin or phospholipids appeared to preserve fibrinogen levels but did not reduce platelet adhesion or consumption.