Mechanical properties of biomimetic tissue adhesive based on the microbial transglutaminase-catalyzed crosslinking of gelatin

Fibrin sealants are a type of soft tissue adhesive that employs biochemical reactions from the late stages of the blood coagulation cascade. Intrinsic to these adhesives are a structural protein and a transglutaminase crosslinking enzyme. We are investigating an alternative biomimetic adhesive based on gelatin and a calcium-independent microbial transglutaminase (mTG). Rheological measurements show that mTG catalyzes the conversion of gelatin solutions into hydrogels, and gel times are on the order of minutes depending on the gelatin type and concentration. Tensile static and dynamic loading of the adhesive hydrogels in bulk form demonstrated that the Young's modulus ranged from 15 to 120 kPa, and these bulk properties were comparable to those reported for hydrogels obtained from fibrin-based sealants. Lap-shear adhesion tests of porcine tissue were performed using a newly published American Society for Testing and Materials (ASTM) standard for tissue adhesives. The gelatin-mTG adhesive bound the opposing tissues together with ultimate adhesive strengths of 12-23 kPa which were significantly higher than the strength observed for fibrin sealants. Even after failure, strands of the gelatin-mTG adhesive remained attached to both of the opposing tissues. These results suggest that gelatin-mTG adhesives may offer the benefits of fibrin sealants without the need for blood products.     

Quantitative characterization of single‐cell adhesion properties by atomic force microscopy using protein‐functionalized microbeads

A method was developed to characterize the adhesion properties of single cells by using protein‐functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin‐functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM‐based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.     

The Use of a Chrome Alum-Gelatin (Subbing) Solution as a General Adhesive for Paraffin Sections

The adhesion obtained from a chrome alum-gelatin solution has been found far superior to results given by widely used general adhesives (Haupt's gelatin and Mayer's egg albumen) for paraffin sections. The subbing solution, which consists of 5.0 gin gelatin and 0.5 gm chrome alum per liter of water, is easier to apply and gives more consistent results. Sections affixed to subbed slides are resistant to removal by acids and bases: 1.0 and 0.1 N HCl or H₂SO₄, 1 M H₃PO₄, 5% oxalic and trichloroacetic acids, 1% and 10% lactic acid, 1.0 and 0.1 N NaOH or NH₄OH, and other fluids and solutions such as organic solvents, water, hypochlorite, KMnO₄ and thiosulfate. The applied adhesive is virtually unstained by many stains, including hematoxylin, eosin, fast green, safranin, PAS, Sudan IV and Mallory's triple stain. The only treatment yet found to detach affixed section in less than 6 hr is immersion in 5% trichloroacetic acid for 15 min at 100 C. The concentration of gelatin and chrome alum in the solution recommended is much lower than in previously described adhesives, but this does not seem to lessen its ability to affix the sections. If the concentrations of gelatin and chrome alum are decreased from those described, adhesive qualities are also decreased. An increase in the concentration of the ingredients causes the adhesive to become stained. The described solution therefore gives optimum adhesion and “resistance” to staining.     

Biocompatibility of a Novel Cyanoacrylate Based Tissue Adhesive: Cytotoxicity and Biochemical Property Evaluation

Cyanoacrylate (CA) is most widely used as a medical and commercial tissue adhesive because of easier wound closure, good cosmetic results and little discomfort. But, CA-based tissue adhesives have some limitations including the release of cytotoxic chemicals during biodegradation. In previous study, we made prepolymerized allyl 2-CA (PACA) based tissue adhesive, resulting in longer chain structure. In this study, we investigated a biocompatibility of PACA as alternative tissue adhesive for medical application, comparing with that of Dermabond® as commercial tissue adhesive. The biocompatibility of PACA was evaluated for short-term (24 hr) and long-term (3 and 7 days) using conventional cytotoxicity (WST, neutral red, LIVE/DEAD and TUNEL) assays, hematoxylin-eosin (H&E) and Masson trichrome (MT) staining. Besides we examined the biochemical changes in cells and DNA induced by PACA and Dermabond® utilizing Raman spectroscopy which could observe the denaturation and conformational changes in protein, as well as disintegration of the DNA/RNA by cell death. In particular, we analyzed Raman spectrum using the multivariate statistical methods including principal component analysis (PCA) and support vector machine (SVM). As a result, PACA and Dermabond® tissue adhesive treated cells and tissues showed no difference of the cell viability values, histological analysis and Raman spectral intensity. Also, the classification analysis by means of PCA-SVM classifier could not discriminate the difference between the PACA and Dermabond® treated cells and DNA. Therefore we suggest that novel PACA might be useful as potential tissue adhesive with effective biocompatibility.     

The effect of a collagen tripeptide fragment (GER) on fibroblast adhesion and spreading depends on properties of an adhesive surface

The effect of collagen tripeptide fragment GER on the adhesion and spreading of mouse embryonic fibroblasts STO to different substrates (polystyrene plastic, poly-L-lysine, fibronectin, gelatin) has been studied. It was found that tripeptide GER was involved in fibroblast adhesion and spreading. The cell response depended both on the mode of tripeptide addition to culture medium and the substrate type. Coincubation of fibroblasts with tripeptide stimulated the cell attachment and spreading to untreated plastic and plastic coated with fibronectin or gelatin but did not change cell adhesion to immobilized poly-L-lysine. Preincubation of cells with tripeptide resulted in partial inhibition of fibroblast adhesion and spreading on fibronectin- and gelatin-coated substrata. It was shown that activation and inhibition of adhesive processes after tripeptide treating was higher on fibronectin than gelatin. The data obtained support the assumption about concerted action of tripeptide GER (activity was dependent both on the used concentration of the tripeptide and the mode of tripeptide addition to culture medium) and chemical characteristics of substrate (polymers of styrene and L-lysine, ECM proteins in native (fibronectin) or partly denatured (gelatin) form) on the cell adhesion and spreading. The main targets that GER peptide may affect during the formation of cell-substrate interactions are discussed.     

Fabrication of agar-gelatin hybrid scaffolds using a novel entrapment method for in vitro tissue engineering applications

Scaffolds of agar and gelatin were developed using a novel entrapment method where agar and gelatin molecules mutually entrapped one another forming stable cell adhesive matrices. Glutaraldehyde was used as a crosslinking agent for gelatin. Three types of hybrid matrices were prepared using agar and gelatin in different proportions in the weight ratio of 1:1, 2:1, and 3:1. Surface characterization of dry scaffolds was carried out by scanning electron microscope. Swelling studies were carried out in phosphate buffer saline (PBS) at physiological pH 7.4. The integral stability of the scaffolds was evaluated by estimating the released disintegrated gelatin from them in PBS at pH 7.4. The attachment kinetics of the cells was evaluated by culturing mouse fibroblast cell line NIH 3T3 on films. The cytocompatibility of these matrices was determined by studying growth kinetics of NIH 3T3 cells on them and morphology of cells was observed through optical photographs taken at various days of culture. It was found that the matrices containing agar and gelatin in 2:1 weight ratio exhibited best growth kinetics. The results obtained from these studies have suggested that the above-described method is a cheap and easy way to fabricate agar-gelatin hybrid scaffolds to grow cells which can be used in various in vitro tissue engineering applications like screening of drugs.     

Design and Characterization of an Adhesive Matrix Based on a Poly(Ethyl Acrylate, Methyl Methacrylate)

The main issue in the development of transdermal patches made of poly(ethyl acrylate, methyl methacrylate) (Eudragit NE 40D, PMM) is the shrinkage phenomenon during the spreading of the latex onto the release liner. To solve this problem, the latex is usually freeze-dried and then re-dissolved in an organic solvent (method 1). To simplify the production process, we prepared an adhesive matrix by adding to the commercial PMM latex a plasticizer and an additive (anti-shrinkage agent) that avoids the shrinkage of the water dispersion spread onto the release liner (method 2). In some cases the active ingredient itself, such as potassium diclofenac (DK) and nicotine (NT), works as anti-shrinkage agent. In this work, the effects of the preparation method, types and concentrations of the plasticizer (triacetin and tributyl citrate) on the adhesive properties of the transdermal patches were investigated. The adhesive properties of the prepared patch were determined by texture analysis, peel adhesion test and shear adhesion. The PMM/plasticizer interactions were evaluated by ATR-FTIR spectroscopy. Furthermore, the in vitro skin permeation profiles of DK and NT released from the patch were determined by Franz cell method. Generally speaking, the variables that mainly modify the adhesive properties are the concentration and type of the plasticizer. The skin permeation profiles of DK and NT from the patch prepared by method 2 overlapped with those obtained with the commercial products. The results underline that the PMM latex can be used conveniently in the development of transdermal patches.     

Do rates of intercellular adhesion measure the cell affinities reflected in cell-sorting and tissue-spreading configurations?

These experiments constitute the first experimental test of the hypothesis that the rates of adhesion between cells measure the intensities of adhesion or tissue affinities that could explain cell sorting and tissue spreading. For any set of relative adhesive intensities between cells in a heterogeneous population, a corresponding minimal free energy configuration can be calculated. This is the cell distribution toward which both cell sorting and tissue spreading should lead. Equilibrium configurations were determined for combinations of 7-day embryonic retina (R) with liver (L) and heart (H), both of which became completely enveloped by R. To produce these results, the adhesive intensities would have to fall in the sequences: L-L > L-R > R-R; and H-H > H-R > R-R. To determine whether the rates of adhesion fall into these same sequences, we have devised a new technique which measures the rates of adhesion between pairs of already-formed cell aggregates of like and unlike kinds. These fall in the sequence L-L > or = H-H > L-H > R-R > H-R > L-R. If these rates paralleled the corresponding intensities of adhesion at configurational equilibrium, both L and H should have become only partially enveloped by R. Thus the rates at which adhesions are initiated do not predict the relative adhesive intensities that could explain the observed tissue configurations.     

壳聚糖基角膜细胞载体的制备及其细胞相容性

为探讨羟丙基壳聚糖基共混膜作为组织工程技术中角膜细胞培养载体的可行性, 分别制备了羟丙基壳聚糖/硫酸软骨素、羟丙基壳聚糖/明胶/硫酸软骨素以及羟丙基壳聚糖/氧化透明质酸/硫酸软骨素三种共混膜。测定其透光率、含水量和蛋白吸附性能; 在共混膜上培养兔角膜上皮细胞, 通过观察角膜上皮细胞在不同载体膜上的生长状态、贴附情况, 测定细胞活性以及上清液中乳酸脱氢酶的活性, 研究三种壳聚糖基载体膜片与角膜上皮细胞的相容性。膜片理化性质测定结果表明三种共混膜片具有良好的透明度, 适宜的含水量和较强的蛋白吸附性能; 细胞相容性实验结果表明羟丙基壳聚糖/明胶/硫酸软骨素共混膜对细胞的损伤最小, 有利于细胞在膜上的贴附和生长, 表现出良好的细胞相容性, 有望作为角膜细胞载体体外构建组织工程化角膜。     

Modulation of spreading, proliferation, and differentiation of human mesenchymal stem cells on gelatin-immobilized poly(L-lactide-co--caprolactone) substrates

Controlled adhesion and continuous growth of human mesenchymal stem cells (hMSCs) are essential for scaffold-based delivery of hMSCs in tissue engineering applications. The main goal of this study is to develop biofunctionalized synthetic substrates to actively control adhesion, spreading, and proliferation of hMSCs. gamma-Ray irradiation was employed to graft acrylic acid (AAc) to biodegeradable poly(L-lactide-co--caprolactone) (PLCL) films. Gelatin, a natural polymer, was then immobilized on the AAc grafted PLCL film (AAc-PLCL) to induce biomimetic interactions with the cells. The graft yield of AAc increased as the irradiation dose and AAc concentration increased, and the presence of gelatin (gelatin-AAc-PLCL) following immobilization was confirmed using ESCA. To investigate cell responses, hMSCs isolated from a human mandible were cultured on the various substrates and their adhesion, spreading, and proliferation were examined. After three days of culture, the DNA concentration from the cells cultured on gelatin-AAc-PLCL film was 2.9-fold greater than that on the PLCL film. Immunofluorescent staining of hMSCs cultured on the gelatin-AAc-PLCL films demonstrated homogeneous localization of F-Actin and vinculin in their cytoplasm, while mature adhesive structure was not observed from the cells cultured on other substrates. Furthermore, the ratio of projected area of adherent single cells on gelatin-AAc-PLCL films was significantly larger (116.80 +/- 12.78%) than that on the PLCL films (30.11 +/- 5.07%). Our results suggest that gelatin-immobilized PLCL substrates may be potentially used in tissue engineering, particularly as a stem cell delivery carrier for the regeneration of target tissue.     

3D-printed PLA/Gel hybrid in liver tissue engineering: Effects of architecture on biological functions

The liver is one of the vital organs in the body, and the gold standard of treatment for liver function impairment is liver transplantation, which poses many challenges. The specific three-dimensional (3D) structure of liver, which significantly impacts the growth and function of its cells, has made biofabrication with the 3D printing of scaffolds suitable for this approach. In this study, to investigate the effect of scaffold geometry on the performance of HepG2 cells, poly-lactic acid (PLA) polymer was used as the input of the fused deposition modeling (FDM) 3D-printing machine. Samples with simple square and bioinspired hexagonal cross-sectional designs were printed. One percent and 2% of gelatin coating were applied to the 3D printed PLA to improve the wettability and surface properties of the scaffold. Scanning electron microscopy pictures were used to analyze the structural properties of PLA–Gel hybrid scaffolds, energy dispersive spectroscopy to investigate the presence of gelatin, water contact angle measurement for wettability, and weight loss for degradation. In vitro tests were performed by culturing HepG2 cells on the scaffold to evaluate the cell adhesion, viability, cytotoxicity, and specific liver functions. Then, high-precision scaffolds were printed and the presence of gelatin was detected. Also, the effect of geometry on cell function was confirmed in viability, adhesion, and functional tests. The albumin and urea production of the Hexagonal PLA scaffold was about 1.22 ± 0.02-fold higher than the square design in 3 days. This study will hopefully advance our understanding of liver tissue engineering toward a promising perspective for liver regeneration.     

Autoradiography of Water-Diffusible Substances in Sections of Whole Baby Rats

Rats, 7 days postnatal which had been injected with a radioactive nuclide, were quick frozen and sectioned in the frozen state. An adhesive cellulose tape (Sellotape) was used to support the section during cutting, through freeze-drying, and attaching to slides. Dehydration of the frozen sections consisted of 1 hr in a chilled desiccator containing silica gel, then at reduced pressure of 2-3 mm Hg until quite dry. The exposed side of the section was sprayed with celloidin dissolved in amyl acetate and allowed to dry. This side of the section was attached to a slide, previously coated with 1% gelatin containing 0.1% chrome alum, by means of an adhesive consisting of 4% gelatin and 5% formalin in 60% glycerol. In applying this adhesive it is mandatory that a border of about 3 mm of bare glass be left outside the adhesive, to allow intimate contact between the sticky side of the tape and the glass. The adhesive was allowed to set for 20 min, the slide immersed in water lor 50 sec, and the cellulose layer of the tape peeled off. The rubber base from the tape was removed with chloroform, the slide dried, and the exposed surface of the section coated with celloidin in amyl acetate, by dipping. After this treatment, the slides could be coated by dipping in autoradiographic emulsion without affecting water-soluble radioactive substances in the tissue.     

Mast cell-fibroblast interactions induce matrix metalloproteinase-9 release from fibroblasts: role for IgE-mediated mast cell activation

Mast cells adhere to fibroblasts, but the biological effects of adhesion are not well understood. We hypothesized that these adhesive interactions are important for tissue remodeling through the release of matrix metalloproteinases (MMP). Murine bone marrow cultured mast cells (BMCMC) were cocultured with NIH-3T3 fibroblasts or murine lung fibroblasts (CCL-206) and supernatants analyzed for MMP-9 release by gelatin zymography. Coculture of BMCMC for 24 h with NIH-3T3 or CCL-206 fibroblasts increased the release of MMP-9 from fibroblasts by 1.7+/-0.2 and 2.0+/-0.7-fold, respectively. Coculture of BMCMC and fibroblasts in the presence of IgE increased further MMP-9 release, which was released by fibroblasts. MMP-9 release was dependent on TNF released from IgE activated BMCMC and on adhesive interactions between BMCMC and fibroblasts. Increased MMP-9 release was also p44/42-dependent, as was MMP-9 up-regulation during coculture of fibroblasts with resting BMCMC. Finally, IgE injection into the mouse ear increased MMP-9 content of the ear tissue in the absence of Ag, indicating that IgE-mediated remodeling may play a pathogenic role in allergic conditions even in the absence of exposure to allergens. In conclusion, mast cell-fibroblast interactions induce the release of proteases important for tissue remodeling, such as MMP-9. MMP-9 release was further increased in the presence of IgE during coculture, suggesting a role for mast cell-fibroblast interactions in atopic conditions.     

Roles of immobilized glycosylated proteins and lipoprotein(a) in adhesivity of endothelial cells: possible implications for atherogenesis.

The adherence of peripheral blood monocytes to adult bovine endothelial cells grown to confluence in the microplates was studied. The microplates were coated with different proteins or used without special treatment. It was shown that endothelial cells seeded on immobilized glycosylated proteins (serum albumin or skin gelatin) adhered more monocytes than the cells grown on non-modified proteins. Endothelial cells grown in lipoprotein(a) coated wells bound more monocytes than the cells grown in non-treated microplates or in wells coated with low density lipoproteins (LDL). The effect of lipoprotein(a) coating could not be reproduced by treating the plates with plasminogen (as a homolog of apo(a)) or with a mixture of LDL and plasminogen. These results indicate that the composition of extracellular substrata has a profound effect on adhesive properties of cultured endothelial cells. The implications of these findings for atherogenesis and for the general aspects of regulation of cell adhesion are discussed.     

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture.     

Micropatterning and characterization of electrospun poly(ε‐caprolactone)/gelatin nanofiber tissue scaffolds by femtosecond laser ablation for tissue engineering applications

Experimental investigations aimed at assessing the effectiveness of femtosecond (FS) laser ablation for creating microscale features on electrospun poly(ε‐caprolactone) (PCL)/gelatin nanofiber tissue scaffold capable of controlling cell distribution are described. Statistical comparisons of the fiber diameter and surface porosity on laser‐machined and as‐spun surface were made and results showed that laser ablation did not change the fiber surface morphology. The minimum feature size that could be created on electrospun nanofiber surfaces by direct‐write ablation was measured over a range of laser pulse energies. The minimum feature size that could be created was limited only by the pore size of the scaffold surface. The chemical states of PCL/gelatin nanofiber surfaces were measured before and after FS laser machining by attenuated total reflectance Fourier transform infrared (ATR‐FTIR) spectroscopy and X‐ray photoelectron spectroscopy (XPS) and showed that laser machining produced no changes in the chemistry of the surface. In vitro, mouse embryonic stem cells (mES cells) were cultured on as‐spun surfaces and in laser‐machined microwells. Cell densities were found to be statistically indistinguishable after 1 and 2 days of growth. Additionally, confocal microscope imaging confirmed that spreading of mES cells cultured within laser‐machined microwells was constrained by the cavity walls, the expected and desired function of these cavities. The geometric constraint caused statistically significant smaller density of cells in microwells after 3 days of growth. It was concluded that FS laser ablation is an effective process for microscale structuring of these electrospun nanofiber tissue scaffold surfaces. Biotechnol. Bioeng. 2011; 108:116–126. © 2010 Wiley Periodicals, Inc.     

Design of a new water-soluble pressure-sensitive adhesive for patch preparation

This work was intended to improve the adhesion properties of an available medical water-soluble pressure-sensitive adhesive (PSA) through the addition of cellulose ethers or polyvinylpyrrolidone (PVP). The adhesion properties were evaluated by means of peel adhesion test and creep resistance test. Possible interactions between the polymethyl methacrylate (PMMA) and hydrocolloid were investigated by Fourier-transformed infrared spectroscopy. Moreover, a central composite design was used to estimate the effects of hydrocolloids and plasticizers and their interactions on the PSA performance. The addition of PVP made it possible to obtain a patch with a 40-fold improved creep compliance and a reduced peel adhesion. The significant increase of the matrix cohesion was due to attractive interactions between the amide group of PVP and the carboxylic acid group of PMMA. The water vapor permeability of the prepared systems was very high. Furthermore, no primary skin irritation was observed. The presence of plasticizers at high level increased both the peel values and creep compliance, showing an opposite behavior with respect to PVP. The new PSA system can be easily removed from the skin, is suitable for repeated applications on the same site, and has adhesive properties that can be modified by changing the component ratios.     

Cellular and molecular approaches to understanding primary adhesion in Enteromorpha: an overview

The attachment of motile spores of the green alga Enteromorpha to the substratum is an active process involving an irreversible commitment to adhesion and the secretion of an adhesive. This paper provides an overview of the spore adhesion processes and outlines the results of an experimental approach towards the molecular characterisation of the adhesive, based on the use of monoclonal antibody (mAb) technology. Hybridomas were produced to settled spores displaying secreted adhesive. Candidates producing mAbs to putative adhesive were selected using a range of criteria based on cellular localisation, time of secretion and functional inhibition of adhesion. MAb Ent 6 immunolabelled fibrillar material which was secreted during the early stages of adhesion and low (nM) concentrations of this mAb, or its F(ab)₂ fragments, strongly inhibited the attachment of zoospores. A related antibody (Ent 1) also labelled the spore adhesive apparatus, but the antigen appeared to be secreted later during the adhesion process and was predominantly associated with the developing cell wall. Ent 1 also inhibited settlement in spore adhesion assays but the effect was most pronounced at later time points which suggests that this antigen does not have a role in the earliest stages of adhesion. Immunolocalisation showed that both antigens were absent from the cytoplasm or organelles of vegetative tissue but labelled the vegetative cell wall, suggesting a relationship between cell wall components and materials involved in primary adhesion. Both mAbs labelled the Golgi region of settled spores, suggesting continued synthesis of both antigens after adhesion. Both mAbs recognised a 110 kDa N‐linked polydisperse and heterogeneous glycoprotein in extracts of swimming spores under denaturing conditions. In native form the antigens behaved as high molecular weight aggregates (M_r>1.3 × 10⁶). The antigens became progressively insoluble after zoospore attachment. Taken together, the data suggest that the two antibodies recognise closely related, polydisperse, self‐aggregating cell wall glycoproteins in which there is some structural variation to suit alternative roles in primary adhesion and cell wall formation. The two mAbs Ent 1 and Ent 6 partially discriminate between these structural and functional variants. A model for zoospore adhesion is discussed in which adhesion is viewed as an extension of cell wall synthesis, with cross‐links between glycoproteins and other cell wall matrix components providing a strong physical continuum between the cell and the adhesive at the substratum interface.     

Pathological activities of Yersinia ruckeri, the Enteric Redmouth (ERM) bacterium

Abstract The adherence and invasive capacities as well as the pathobiological activities exhibited by Yersinia ruckeri were examined. Although adhesive ability was dependent on the cell-line employed, all the strains showed moderate adhesion and invasiveness in the salmon cell-line CHSE-214. With regard to the extracellular products (ECP) all of them were strongly toxic for fish with LD₅₀ ranging from 2 to 9.12 μg protein per g fish. In addition, all the ECP samples showed caseinase, gelatinase, amylase, lipase and phospholipase activities, hydrolysed esculin and displayed hemolytic activities for trout, salmon, sheep and human erythrocytes. Heat treatment (100°C for 10 min) caused the loss of all these biological activities except the hydrolysis of gelatin. On the other hand, SDS-PAGE analysis of the LPS and protein components of the ECP revealed variations among strains depending on the serotype. The lack of lethal effects of the LPS present in the ECP was also demonstrated.