生物材料的表面修饰对细胞黏附性的影响

细胞与生物材料相互作用过程中,细胞黏附是首先发生的生物学行为,对后续发生的迁徙、增殖和分化具有重要影响.因此,如何改善细胞在生物材料表面的黏附特性,自然成为目前组织工程研究的一项重要内容,目前较多采用的是对生物材料进行表面修饰的方法.就生物材料的表面修饰对细胞在生物材料上黏附的影响进展作一综述.     

RGD modified polymers: biomaterials for stimulated cell adhesion and beyond

Since RGD peptides (R: arginine; G: glycine; D: aspartic acid) have been found to promote cell adhesion in 1984 (Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule, Nature 309 (1984) 30), numerous materials have been RGD functionalized for academic studies or medical applications. This review gives an overview of RGD modified polymers, that have been used for cell adhesion, and provides information about technical aspects of RGD immobilization on polymers. The impacts of RGD peptide surface density, spatial arrangement as well as integrin affinity and selectivity on cell responses like adhesion and migration are discussed.     

Cell responses to biomaterials. II: Endothelial cell adhesion and growth on perfluorosulfonic acid

We report here the use of perfluorosulfonic acid (Nafion) as a substratum for the growth of bovine aortal endothelial cells. This support which can be generated in a number of forms is at least as efficient in maintaining the growth of endothelial and other cell types as tissue culture grade polystyrene (TCP) and represents an advance in this regard over polytetrafluoroethylene (Teflon). The mechanism underlying the different cell attachment capacities of these three polymers is not readily related to their different protein binding patterns. While Nafion adsorbs more total protein from serum than Teflon or TCP, it adsorbs relatively less of the major cell adhesive proteins, vitronectin and fibronectin, than does Teflon. Both Nafion and Teflon had comparable but low thrombogenic potential by in vitro tests. Teflon or expanded Teflon (Gore-tex) coated with a thin film of Nafion assumes the cell supportive characteristics of Nafion and hence the modification of these surfaces by the induction of a stable bond between Teflon (in various forms) and Nafion may provide a composite vascular graft material which has all the desirable qualities of both materials.     

Sub-micron texturing for reducing platelet adhesion to polyurethane biomaterials

Platelet adhesion is a key event in thrombus development on blood-contacting medical devices. It has been demonstrated that changes to the chemistry of a material surface can reduce platelet adhesion. In this work, it is hypothesized that sub-micron surface textures may also reduce adhesion via a decrease in the surface area of material with which platelets can make contact, and hence a decreased probability of interaction with adhesive ligands. A polyether(urethane urea) was textured with two different sizes of sub-micron pillars using a replication molding technique that did not alter the material surface chemistry. Adhesion of platelets was assessed in a physiologically relevant shear stress range of 0-67 dyn/cm2 using a rotating disk system. Platelets were immunofluorescently labeled and adhesion was compared on smooth and textured samples. Platelet adhesion was greatest at low shear stress ranging from 0 to 5 dyn/cm2, and sub-micron textures were observed to reduce platelet adhesion in this range. Additionally, non-adherent platelets did not demonstrate large-scale activation after exposure to textured samples. We conclude that surface textures with sub-platelet dimensions may reduce platelet adhesion from plasma to polyether(urethane urea) at low shear stress.     

An improved image analysis method for cell counting lends credibility to the prognostic significance of T cells in colorectal cancer

Numerous immunohistochemically detectable proteins, such as immune cell surface (CD) proteins, vascular endothelial growth factor, and matrix metalloproteinases, have been proposed as potential prognostic markers in colorectal cancer (CRC) and other malignancies. However, the lack of reproducibility has been a major problem in validating the clinical use of such markers, and this has been attributed to insufficiently robust methods used in immunohistochemical staining or its assessment. In this study, we assessed how computer-assisted image analysis might contribute to the reliable assessment of positive area percentage and immune cell density in CRC specimens, and subsequently, we applied the computer-assisted cell counting method in assessing the prognostic value of T cell infiltration in CRC. The computer-assisted analysis methods were based on separating hematoxylin and diaminobenzidine color layers and then applying a brightness threshold using open source image analysis software ImageJ. We found that computer-based analysis results in a more reproducible assessment of the immune positive area percentage than visual semiquantitative estimation. Computer-assisted immune cell counting was rapid to perform and accurate (Pearson r > 0.96 with exact manual cell counts). Moreover, the computer-assisted determination of peritumoral and stromal T cell density had independent prognostic value. Our results suggest that computer-assisted image analysis, utilizing freely available image analysis software, provides a valuable alternative to semiquantitative assessment of immunohistochemical results in cancer research, as well as in clinical practice. The advantages of using computer-assisted analysis include objectivity, accuracy, reproducibility, and time efficiency. This study supports the prognostic value of assessing T cell infiltration in CRC.     

An image analysis strategy to determine the distribution of cell types in spleen sections.

An image analysis strategy was designed to objectively determine distribution patterns of cell types in spleen sections. The strategy was applied to rat spleen cryostat sections that were strained immunohistochemically by means of monoclonal antibodies against different populations of lymphocytes and macrophages. The strategy revealed three segments of the spleen beginning in the middle of a central arteriole and ending within the red pulp. In each of these segments, three consecutive zones were established: the white pulp, the marginal zone, and the red pulp. In each tissue section, three segments were selected starting in two different arterioles. Consequently, six segments were analysed in each section. Special software was used to calculate percentages of positive staining in all zones in each segment. Image analysis data for each monoclonal antibody tested correlated closely with microscopical observations. The proposed strategy allows objective quantification of lymphocyte and macrophage populations and their distribution patterns. It is an useful tool for studying imbalances in cell populations in the spleen due to immune challenges.     

Complement activation mediates cellular adhesion to synthetic biomaterials

The possibility has arisen that it is the complement proteins of blood plasma that mediates cellular adhesion following the exposure of a synthetic material to blood. There are two means of investigating this possibility. One is by pharmacologically rendering the complement system of an animal incapable of being activated before its blood is exposed to a material and determining the effect on the degree of cellular adhesion that results. The second is to leave the hemostasis system fully in tact but to modify the material so that the material activates less complement when it is exposed to blood and to determine if this also reduces the degree of cellular adhesion. We review the results of a series of studies that involve both of these approaches. The evidence from both indicate that the complement system mediates cellular adhesion to synthetic materials.     

Crosslinked gelatin beads: A culture method for the study of cell adhesion and invasion

Summary A method is described for culturing invasive cell lines on crosslinked gelatin beads and preparing them for immunocytochemical and morphological observation. Very invasive cells such as Rous sarcoma virus-transformed chicken embryo fibroblasts, and human melanoma LOX and RPMI7951 and breast carcinoma MDA-MB-231 cells will actively degrade this matrix, extending cellular protrusions, called invadopodia, into the sites of degradation. Normal chicken embryo fibroblasts and other non-invasive cell lines do not disrupt the surface of these beads and do not form invadopodia. Invadopodia extending into the bead can be visualized by electron microscopy. Cellular removal of fluorescent fibronectin that has been covalently coupled to the bead surface can be monitored using fluorescence microscopy of frozen-thin-sections. In double label experiments, immunocytochemistry is used to localize antigens in invadopodia at sites of membrane invasion. The materials for bead preparation are inexpensive, and this method has the advantage that many cell types will attach and spread readily on the beads, while only highly invasive cells will invade into the bead.     

An image analysis study of adenovirus type 5-induced crystalline inclusions.

A combined electron microscopic and optical diffractometric study of Ad5-induced non-virion protein crystals found in the nuclei of infected KB cells at late times (48 to 72 h p.i.) after infection has been carried out. Data obtained have indicated that the unit cell of the crystal is rectangular and not hexagonal as previously presumed.     

Inhibition by heparin and derivatized dextrans of Staphylococcus aureus adhesion to fibronectin-coated biomaterials.

Recent data on cardiovascular device-centered infections suggest that some plasma and extracellular matrix proteins contribute to bacterial adhesion and colonization on biomaterials. We previously developed an in vitro assay to study the Staphylococcus aureus adhesion-promoting effect of surface-adsorbed fibronectin on flat PMMA coverslips coated with a monolayer amount of fibronectin. We screened the potential anti-adhesive properties of a group of substituted dextrans, previously shown to exhibit potent anticoagulant and anticomplementary activities. In comparison to unsubstituted dextran which showed no significant (< 20%) adhesion inhibition at 1 mg/ml, dextrans increasingly substituted with carboxylic and benzylamide groups (CMBD) exhibited increasing anti-adhesive activities. Three CMBD derivatives showing an increasing proportion (5-14%) of benzylamide groups showed inhibition of bacterial adhesion increasing from 33 to 51% at 1 mg/ml. Another category of substituted dextrans having a variable proportion (2-26%) of sulfonated benzylamide groups (CMBDS) produced active inhibition of S. aureus adhesion. In comparison to these heparin-like dextran derivatives, native heparin produced inhibition values of S. aureus adhesion which were intermediate between those of CMBD and CMBDS compounds. Furthermore, the anti-adhesive activity was still expressed when substituted dextrans were preincubated with fibronectin-coated PMMA but washed away at the time when radiolabeled bacteria were added to the adhesion assay. This indicates that the anti-adhesive effects of CMBDS could be exerted at the level of the S. aureus binding site of fibronectin. In conclusion, S. aureus adhesion on fibronectin-coated biomaterials can be efficiently blocked in vitro by soluble compounds such as dextran derivatives.     

Comparison of bacterial and tissue cell initial adhesion on hydrophilic/hydrophobic biomaterials

In this study, interactions of widely-used polymeric biomaterials, i.e. poly(hydroxyethyl methacrylate) (PHEMA) and its copolymer with dimethylaminoethyl methacrylate (PHEMA-20% DMAEMA), polyurethane (PU), polypropylene (PP), poly(vinyl chloride) (PVC), and poly(lactide-glycolide) (PLGA), with three pathogenic bacteria and one nonpathogen were investigated comparatively with the adhesion of two tissue cells in different morphologies, i.e. fibroblast-like baby hamster kidney (BHK 21) cells and epithelial Madine Darby kidney (MDBK) cells. Biomaterials were prepared in the membrane form by bulk polymerization or solvent casting. Surface characterization studies showed that these polymers have different surface free energies in the range of 26.9-63.1 erg cm(-2) and they have smooth surfaces. The bacteria used were; Escherichia coli ATCC 25922, Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus, and Lactobacillus acidophilus B-13. Initial adhesion of bacteria to the polymeric surfaces was examined under static conditions and in a laminar flow cell. The adhesion behaviour of S. aureus and S. epidermidis was found independent of the polymeric surface hydrophobicity. However, the percentage of attached E. coli decreased when increasing the surface free energy of the polymer, while L. acidophilus showed just the opposite behaviour. The comparative results indicated that the adhesion of BHK and MDBK cell was lowest on the most hydrophilic PHEMA surface and highest on the most hydrophobic PP surface. In contrast to the case of bacterial adhesion, no relationship was found between polymer hydrophobicity and mammalian cell adherence.     

Mast cell adhesion to fibronectin.

The MCP-5 murine mast cell line, as well as primary bone marrow-derived cultured mast cells (BMCMC), are demonstrated to bind to fibronectin, a ubiquitous adhesion protein of the extracellular matrix. BMCMC required activation by phorbol myristate acetate (PMA) to adhere to fibronectin, whereas MCP-5 displayed spontaneous adherence. The binding of both MCP-5 and BMCMC was dose dependent, with maximal adhesion at a fibronectin concentration of 20 micrograms/ml. The 120,000 molecular weight (MW) proteolytic fragment of fibronectin containing the RGDS cell attachment site was able to substitute for the native fibronectin molecule in promoting mast cell attachment. Mast cell adhesion to fibronectin, in addition, could be inhibited by the RGDS peptide alone. These data suggest that, in addition to the previously described mast cell-laminin interactions, mast cells also adhere to fibronectin, thus providing further insight into their tissue localization and possible roles in processes such as wound healing and fibrosis.     

抗菌生物材料的抗细菌黏附能力

背景：由于生物材料和人工器官在临床应用逐渐增多,给临床患者进行治疗疾病的同时还存在着一些问题,最为常见的是生物材料植入人体后引起的细菌感染。 目的：探讨生物材料在抗细菌黏附中的作用,抗菌生物材料的分类及特点。 方法：生物材料在机体引起各种感染的原因是由于细菌生物膜的形成,防止生物材料置入后感染的关键是抑制细菌在生物材料表面的黏附以及防止细菌在生物材料表面形成细菌生物膜。细菌表面黏附重点是改变细菌自身的特性和材料表面的物理化学性质,通过改变材料的物理化学性质来减小材料和细菌之间的相互作用力,主要采用化学接枝法、等离子体法、气相沉淀法等。预防细菌黏附首先要增强机体的免疫防御能力,其次要使界面快速的被组织覆盖,形成严密的连结界面。 结果与结论：抗菌生物材料分为无机抗菌生物材料、天然抗菌生物材料和合成抗菌生物材料,无机抗菌材料以银系材料为主,天然抗菌生物材料以壳聚糖研究为较多,合成抗菌生物材料以季铵盐类材料为代表,各种材料都具有各自的优缺点,需要进一步的体内外基础实验和临床研究来验证和推动抗菌生物材料的发展。     

An image analysis study on nuclear morphology in metastasized and non-metastasized squamous cell carcinomas of the tongue

In a restrospective case–control study on 46 metastasized and 34 non-metastasized primary tongue carcinomas, the nuclear morphology and chromatin pattern were assessed in 3 μm thick, formalin-fixed, paraffin-embedded, and Feulgen-stained tissue sections of surgical resection specimens, by means of high-resolution computer-assisted image analysis. The aim of this study was to disclose differences in karyometric features, such as nuclear size-, shape-, and chromatin-pattern features, between these groups, with a view to developing a discriminant function that can predict the occurrence of metastasis for the individual patient. In addition, the lymph node metastases of 31 patients and the normal tongue epithelium of 21 patients were also assessed, to study the possible differences between these two groups and primary tumours. In the metastasized tumours, the chromatin was significantly more condensed (P=0·01) and exhibited significantly less variation in chromatin condensation (P<0·001) than in the group of non-metastasized carcinomas. Comparison of lymph node metastases with their primary tumours disclosed only minor differences in chromatin pattern. These findings suggest that only minor genetic differences exist between primary tongue carcinomas and their metastases. Tumour cells of tongue carcinomas showed highly significant differences from cells of normal tongue mucosa for most karyometric features. Logistic regression analysis resulted in a classifier, based on the circularity of the nucleus (CIRC ) and the standard deviation of the chromatin condensation (SD COND ), to predict the occurrence of lymph node metastases. After cross-validation, the percentages of correct classifications in the group of metastasized and non-metastasized tumours were 72 and 62 per cent, respectively. These results are comparable to the classification results obtained from a classifier based on the clinical T-stage, but our karyometric classification results show a much more equal distribution between the sensitivity and specificity. Karyometric features appeared to be more appropriate to predict metastates than biomarkers such as p53, bcl-2, and Ki-67. © 1998 John Wiley & Sons, Ltd.     

血浆蛋白对生物材料细菌粘附影响研究进展

防止生物材料细菌粘附是防治生物材料为中心感染(biomaterial centered infect,BCI)的重要环节.研究发现,人体血浆蛋白对生物材料细菌粘附有重要影响.因此,研究血浆蛋白与生物材料细菌粘附关系为防治BCI有重要的意义.本文综述了与生物材料细菌粘附相关的血浆蛋白、血浆蛋白对生物材料细菌粘附影响的有关机制及如何提高血浆蛋白抗细菌粘附作用的展望.     

The influence of thrombus components in mediating bacterial adhesion to biomaterials

Thrombosis and infection represent the two largest limiting factors determining the long term success of implanted biomaterials. Infections associated with biomaterials are difficult to treat, and appear to evade the host defense systems. Mechanisms relating infection to thrombosis are described. Investigations into the role of receptors in mediating adhesion to thrombi are also discussed, in addition to strategies to reduce bacterial adhesion to biomaterial surfaces.     

PEG-variant biomaterials as selectively adhesive protein templates: model surfaces for controlled cell adhesion and migration

Our study focused on the role of poly(ethylene glycol) (PEG) in actively regulating the biological responsiveness of protein-adsorbed biomaterials. To this end, we designed PEG-variant biomaterials from a family of tyrosine/PEG-derived polycarbonates to present surfaces ranging from low to intermediate levels of PEG concentration, below the PEG level requisite for complete abolition of protein adsorption. We analyzed the effect of PEG concentration on the amount, conformation and bioactivity of an adsorbed model protein, fibronectin, and on the attachment, adhesion strength and motility of L929 fibroblasts. Our results demonstrate that low levels of PEG can regulate not only the extent but also the conformation and specific bioactivity of adsorbed fibronectin. As the PEG concentration was increased from 0 to 6 mol%, the amount of adsorbed fibronectin decreased linearly yet the fibronectin conformation was altered such that the overall bioactivity of adsorbed fibronectin was uncompromised. We report that the degree of cell attachment varied with PEG concentration in a manner similar to the dependence of fibronectin bioactivity on PEG. In contrast, the nature of cell adhesion strength dependence on PEG paralleled the pattern observed for fibronectin surface concentration. Our studies also indicated that the rate of cell migration was inversely correlated with PEG concentration over a narrow range of PEG concentration. Overall, these results highlight the striking ability of PEG-variant biomaterials to systematically regulate the behavior of adsorbed cell adhesion proteins and, consequently, effect cell functions.