Novel surface modification of three-dimensional bacterial nanocellulose with cell-derived adhesion proteins for soft tissue engineering.

Bacterial nanocellulose (BNC) is a natural polymer composed of glucose units with an important application as a two and three-dimensional scaffold for tissue engineering. However, as a polysaccharide, BNC does not have the biological signals of protein biomaterials. Therefore, this paper aims to develop a novel methodology to biomimic soft extracellular matrix (ECM) chemistry on to 3D BNC using the bioengineering of fibroblasts (the cells responsible for producing and regenerating the ECM) to immobilise adhesion proteins such as collagen and fibronectin. Modified 3D BNC (Mod-BNC) biomaterials were morphologically, thermally, and chemically characterised, and furthermore, the cell response was analysed by adhesion studies using atomic force microscopy (AFM), XTT assay, and confocal microscopy. Cell-derived proteins were deposited on the BNC nanoribbon network to modify its surface. The contact angle was increased from 40° to 60°, reducing the wettability of the biomaterial, and during thermogravimetry, the proteins in Mod-BNC exhibited an enhanced thermal stability because of the interactions between themselves and BNC. Chemical and immunocytochemistry analyses confirmed the presence of collagen type I and fibronectin on 3D BNC. These proteins activate integrin adhesion pathways that generate stronger cell adhesions. AFM experiments showed higher forces and energies on modified biomaterials, and moreover, the cells that adhered on to Mod-BNC exhibited higher mitochondrial activity and higher cell populations per cubic millimetre than non-modified surfaces (NMod-BNC). Accordingly, it was established that this novel methodology is robust and able to biomimic the chemical surface of soft ECM and immobilise cell-derived adhesion proteins from fibroblast; moreover, the Mod-BNC exhibited better cell response than NMod-BNC because of the biological signals in 3D BNC.

Current Developments in Antimicrobial Surface Coatings for Biomedical Applications.

Bacterial adhesion and subsequent biofilm formation on material surfaces represent a serious problem in society from both an economical and health perspective. Surface coating approaches to prevent bacterial adhesion and biofilm formation are of increased importance due to the increasing prevalence of antibiotic resistant bacterial strains. Effective antimicrobial surface coatings can be based on an anti-adhesive principle that prevents bacteria to adhere, or on bactericidal strategies, killing organisms either before or after contact is made with the surface. Many strategies, however, implement a multifunctional approach that incorporates both of these mechanisms. For anti-adhesive strategies, the use of polymer chains, or hydrogels is preferred, although recently a new class of super-hydrophobic surfaces has been described which demonstrate improved anti-adhesive activity. In addition, bacterial killing can be achieved using antimicrobial peptides, antibiotics, chitosan or enzymes directly bound, tethered through spacer-molecules or encased in biodegradable matrices, nanoparticles and quaternary ammonium compounds. Notwithstanding the ubiquitous nature of the problem of microbial colonization of material surfaces, this review focuses on the recent developments in antimicrobial surface coatings with respect to biomaterial implants and devices. In this biomedical arena, to rank the different coating strategies in order of increasing efficacy is impossible, since this depends on the clinical application aimed for and whether expectations are short- or long term. Considering that the era of antibiotics to control infectious biofilms will eventually come to an end, the future for biofilm control on biomaterial implants and devices is likely with surface-associated modifications that are non-antibiotic related.

An in vivo study of composite microgels based on hyaluronic acid and gelatin for the reconstruction of surgically injured rat vocal folds.

PURPOSE The objective of this study was to investigate local injection with a hierarchically microstructured hyaluronic acid-gelatin (HA-Ge) hydrogel for the treatment of acute vocal fold injury using a rat model. METHOD Vocal fold stripping was performed unilaterally in 108 Sprague-Dawley rats. A volume of 25 µl saline (placebo controls), HA-bulk, or HA-Ge hydrogel was injected into the lamina propria (LP) 5 days after surgery. The vocal folds were harvested at 3, 14, and 28 days after injection and analyzed using hematoxylin and eosin staining and immunohistochemistry staining for macrophages, myofibroblasts, elastin, collagen type I, and collagen type III. RESULTS The macrophage count was statistically significantly lower in the HA-Ge group than in the saline group (p < .05) at Day 28. Results suggested that the HA-Ge injection did not induce inflammatory or rejection response. Myofibroblast counts and elastin were statistically insignificant across treatment groups at all time points. Increased elastin deposition was qualitatively observed in both HA groups from Day 3 to Day 28, and not in the saline group. Significantly more elastin was observed in the HA-bulk group than in the uninjured group at Day 28. Significantly more collagen type I was observed in the HA-bulk and HA-Ge groups than in the saline group (p < .05) at Day 28. The collagen type I concentration in the HA-Ge and saline groups was found to be comparable to that in the uninjured controls at Day 28. The concentration of collagen type III in all treatment groups was similar to that in uninjured controls at Day 28. CONCLUSION Local HA-Ge and HA-bulk injections for acute injured vocal folds were biocompatible and did not induce adverse response.

Poly(trimethylene carbonate) and biphasic calcium phosphate composites for orbital floor reconstruction: a feasibility study in sheep.

In the treatment of orbital floor fractures, bone is ideally regenerated. The materials currently used for orbital floor reconstruction do not lead to the regeneration of bone. Our objective was to render polymeric materials based on poly(trimethylene carbonate) (PTMC) osteoinductive, and to evaluate their suitability for use in orbital floor reconstruction. For this purpose, osteoinductive biphasic calcium phosphate (BCP) particles were introduced into a polymeric PTMC matrix. Composite sheets containing 50 wt% BCP particles were prepared. Also laminates with poly(D,L-lactide) (PDLLA) were prepared by compression moulding PDLLA films onto the composite sheets. After sterilisation by gamma irradiation, the sheets were used to reconstruct surgically-created orbital floor defects in sheep. The bone inducing potential of the different implants was assessed upon intramuscular implantation. The performance of the implants in orbital floor reconstruction was assessed by cone beam computed tomography (CBCT). Histological evaluation revealed that in the orbital and intramuscular implantations of BCP containing specimens, bone formation could be seen after 3 and 9 months. Analysis of the CBCT scans showed that the composite PTMC sheets and the laminated composite sheets performed well in orbital floor reconstruction. It is concluded that PTMC/BCP composites and PTMC/BCP composites laminated with PDLLA have osteoinductive properties and seem suitable for use in orbital floor reconstruction.

In vivo behaviour of a biodegradable poly(trimethylene carbonate) barrier membrane: a histological study in rats.

The aim of the present study was to evaluate the response of surrounding tissues to newly developed poly(trimethylene carbonate) (PTMC) membranes. Furthermore, the tissue formation beneath and the space maintaining properties of the PTMC membrane were evaluated. Results were compared with a collagen membrane (Geistlich BioGide), which served as control. Single-sided standardized 5.0 mm circular bicortical defects were created in the mandibular angle of rats. Defects were covered with either the PTMC membrane or a collagen membrane. After 2, 4 and 12 weeks rats were sacrificed and histology was performed. The PTMC membranes induced a mild tissue reaction corresponding to a normal foreign body reaction. The PTMC membranes showed minimal cellular capsule formation and showed signs of a surface erosion process. Bone tissue formed beneath the PTMC membranes comparable to that beneath the collagen membranes. The space maintaining properties of the PTMC membranes were superior to those of the collagen membrane. Newly developed PTMC membranes can be used with success as barrier membranes in critical size rat mandibular defects.

A comparative study on the viscoelastic properties of human and animal lenses.

A new method of compression between two parallel plates is used to measure the viscoelastic properties of whole and decapsulated human lenses and compare them with other animal species. Compressive load relaxation was performed by deforming the lens by 10% and measuring the force relaxation response for 100 s to obtain thickness, stiffness and relaxation of the induced loading force and Maxwell parameters for human, monkey, porcine and leporine whole and decapsulated lenses. Thickness and percentage loading force relaxation increased linearly with lens age, whereas stiffness and induced loading force increased exponentially. Human and monkey lenses aged at different rates. Loading force relaxation in a generalized Maxwell model was described by three time constants ranging from 1 to 1000 s. Compressive load relaxation is a very versatile method to study the viscoelastic properties of whole and decapsulated lenses and potentially also artificial accommodating lenses. The data presented in the study will help researchers choose the most suitable animal lenses based on the desired properties and age to be mimicked from the human lenses.

In vitro degradation of a biodegradable polyurethane foam, based on 1,4-butanediisocyanate: a three-year study at physiological and elevated temperature.

Biodegradable polyesterurethanes (PUs) may be used as scaffold materials for tissue regeneration applications, because of their excellent mechanical properties. In this study, the degradation of highly porous PU foams was evaluated in vitro. The PU had amorphous soft segments of DL-lactide/epsilon-caprolactone and uniform hard segments, synthesized from 1,4-butanediisocyanate and butanediol. The foams were degraded for 3 years in a Sörensen buffer solution (pH 7.4) at 37 and 60 degrees C. Dimensions of the foams, intrinsic viscosity, mass loss, thermal properties, and composition of the remaining material were evaluated. Copolyester (CP) foams of DL-lactide/epsilon-caprolactone served as controls. The PU foams kept their dimensions for 20 weeks at 37 degrees C, whereas CP foams collapsed after 3 weeks. PU mass loss reached a maximum of 80% at both 37 and 60 degrees C. CP mass loss reached 99.9% at 60 degrees , and 92% at 37 degrees C after 3 years. The degradation processes at 37 and 60 degrees C are initially the same, but eventually degradation products with different thermal properties are being formed. (1)H NMR studies showed that the hard urethane segments of the PU do not degrade in vitro at pH 7.4. It was concluded that the PU material has favorable characteristics for a scaffold material. Compared to long-term in vivo results of the same PU these in vitro results are not representative for the in vivo situation and therefore total resorption has to be investigated in long-term in vivo studies.

In vivo resorption of a biodegradable polyurethane foam, based on 1,4-butanediisocyanate: a three-year subcutaneous implantation study.

Degradable polyurethanes (PUs), based on aliphatic diisocyanates, can be very useful in tissue regeneration applications. Their long-term in vivo degradation has not been extensively investigated. In this study a biodegradable PU with copolyester soft segments of DL-lactide/epsilon-caprolactone and hard segments synthesized from 1,4-butanediisocyanate was evaluated with regard to tissue response during degradation and, ultimately, the resorption of the material. Highly porous PU foam discs were subcutaneously implanted in rats and rabbits for intervals up to 3 years. A copolymer foam of DL-lactide and epsilon-caprolactone served as a control. The foams, the surrounding tissues and the draining lymph nodes were evaluated with light and electron microscopy. In the first stages of degradation the number of macrophages and giant cells increased. As the resorption stage set in their numbers gradually decreased. Electron microscopy showed macrophages containing pieces of PU. The size of the intracellular PU particles diminished and cells containing these remnants gradually disappeared after periods from 1 to 3 years. After 3 years an occasional, isolated macrophage with biomaterial remnants could be traced in both PU and copolymer explants. Single macrophages with biomaterial remnants were observed in the lymph nodes between 39 weeks and 1.5 years following implantation. It is concluded that the PU foam is biocompatible during degradation. After 3 years PU samples had been resorbed almost completely. These results indicate that the PU foam can be safely used as a biodegradable implant.

A long-term in vitro biocompatibility study of a biodegradable polyurethane and its degradation products.

The biological safety of degradation products from degradable biomaterials is very important. In this study a new method is proposed to test the cytotoxicity of these degradation products with the aim to save time, laboratory animals, and research funds. A biodegradable polyurethane (PU) foam was subjected to this test method. The PU had soft segments of DL-lactide/epsilon-caprolactone and hard segments synthesized from butanediol and 1,4-butanediiosocyanate. Copolymer foams without urethane segments, consisting of DL-lactide/epsilon-caprolactone, were tested as well. Accumulated degradation products were collected by degrading the foams in distilled water at 60 degrees C up to 52 weeks. Cell-culture medium was prepared from powder medium with this water. In different tests the cytotoxicity of this medium was established. The first signs of cytotoxicity were observed after 3-5 weeks of degradation. This accounts for both materials and reestablishes the good short-term biocompatibility of these materials. The PU showed more toxicity toward the end stages of degradation in comparison with the copolymer. This is probably related to the accumulation of degradation products of the urethane segments. In the in vivo situation the degradation of the PU and the metabolism and excretion of degradation products may differ. Therefore, long-term in vivo studies will have to establish whether these in vitro results are representative for the in vivo behavior of the degrading PU.

An animal model for oroantral communications: a pilot study with Göttingen minipigs.

A pilot study was performed to investigate whether the Göttingen minipig is a suitable animal model for creating and closing oroantral communications (OACs) and to test whether these defects can be closed with a biodegradable polyurethane (PU) foam. In three adult minipigs, an OAC was created on both sides of the maxilla. The left side was closed by a standard surgical buccal flap procedure, the right side by applying a PU foam. The pigs were killed after two weeks, one month and three months, respectively. Postmortem and histological examination showed that an OAC was created in only one of six cases. In the remaining cases, the infraorbital canal was perforated instead of the floor of the maxillary sinus. It was concluded that the Göttingen minipig is not a suitable animal model for OAC investigations. As a result, the closure of OACs with a biodegradable PU could not be evaluated.

Short-term in vitro and in vivo biocompatibility of a biodegradable polyurethane foam based on 1,4-butanediisocyanate.

In this study short-term in vitro and in vivo biocompatibility apects of a biodegradable polyurethane (PU) foam were evaluated. The PU consists of hard urethane segments and amorphous soft segments based on a copolyester of dl-lactide and epsilon-caprolactone. The urethane segments are of uniform length and synthesized with 1,4-butanediisocyanate. The foam has good mechanical properties and will be used for tissue regeneration applications. Degradation tests were carried out in a buffer solution for twelve weeks. Cytotoxicity was determined using extract and direct contact test methods with incubation periods varying form 24 to 72 h. The foam was implanted subcutaneously for one, four and twelve weeks and the tissue response to the material was histologically evaluated. In vitro, the mass loss was 3.4% after twelve weeks. In the cytotoxicity tests the PU caused no abnormal growth behaviour, nor morphological changes or inhibition in metabolic activity. The in vivo studies showed no toxic tissue response to the PU. Connective tissue ingrowth, accompanied by vascular ingrowth was complete at twelve weeks. In vivo degradation had started within four to twelve weeks. In conclusion, the PU shows a good in vitro and in vivo biocompatibility in these short-term experiments.

The use of hydrophobins to functionalize surfaces.

The physiochemical nature of surfaces can be changed by small proteins which are secreted by filamentous fungi. These proteins, called hydrophobins, are characterized by the presence of eight conserved cysteine residues and a typical hydropathy pattern. Upon contact with a hydrophilic-hydrophobic interface they self-assemble into highly insoluble amphipathic membranes. As a result, hydrophobic surfaces become hydrophilic and vice versa. Genetic engineering of hydrophobins was used to study structure-function relationships. In addition, engineered hydrophobins were constructed to increase the biocompatibility of surfaces. The glycosylated N-terminal region of the mature SC3 hydrophobin was deleted and the cell-binding domain of human fibronectin was introduced at the N-terminus. The gross properties of the hydrophobins were not affected. However, the physiochemical properties of the hydrophilic side of the assembled protein did change. Growth of fibroblasts on Teflon could be improved by coating the solid with the engineered hydrophobins. Thus, by changing the N-terminal part of hydrophobins, the physiochemical nature of the hydrophilic side of the assembled form can be altered and a variety of new functionalities introduced. The fact that hydrophobins self-assemble at any hydrophilic-hydrophobic interface, irrespective of the chemical nature of the surface, therefore provides a generic approach to modify surfaces and make them interesting candidates for the use in various technical and medical applications.

Promotion of fibroblast activity by coating with hydrophobins in the beta-sheet end state.

Hydrophobins such as SC3 and SC4 of Schizophyllum commune self-assemble into an amphipathic film at hydrophilic/hydrophobic interfaces. These proteins can thus change the nature of surfaces, which makes them attractive candidates to improve physio- and physico-chemical properties of implant surfaces. At a hydrophobic solid, assembly of the hydrophobin is arrested in an intermediate state, called the alpha-helical state. The conversion to the stable beta-sheet end state can be induced by treating the solid at elevated temperatures in the presence of detergent. We here show that SC3 and SC4 in the alpha-helical state homogeneously cover Teflon sheets when coating was performed at 20 degrees C. However, when the protein was adsorbed at 80 degrees C aggregates were shown to bind tightly to the adsorbed hydrophobin film. The transition to the beta-sheet state created pores of about 50 nm in the SC3 and SC4 coatings when coating was performed at 20 degrees C. Cell growth and morphology on SC4 coatings was better than on SC3. In case of both hydrophobins, fibroblast growth and morphology was not influenced by the coating temperature or the conformation of the protein. However, in contrast to the alpha-helical state, the beta-sheet state of both SC3 and SC4 hardly, if at all, affected mitochondrial activity.

Animal models for tracheal research.

Tracheal research covers two main areas of interest: tracheal reconstruction and tracheal fixation. Tracheal reconstructions are aimed at rearranging or replacing parts of the tracheal tissue using implantation and transplantation techniques. The indications for tracheal reconstruction are numerous: obstructing tracheal tumors, trauma, post-intubation tissue reactions, etc. Although in the past years much progress has been made, none of the new developed techniques have resulted in clinical application at large scale. Tissue engineering is believed to be the technique to provide a solution for reconstruction of tracheal defects. Although developing functional tracheal tissue from different cultured cell types is still a challenge. Tracheal fixation research is relatively new in the field and concentrates on solving fixation-related problems for laryngectomized patients. In prosthetic voice rehabilitation tracheo-esophageal silicon rubber speech valves and tracheostoma valves are used. This is often accompanied by many complications. The animal models used for tracheal research vary widely and in most publications proper scientific arguments for animal selection are never mentioned. It showed that the choice on animal models is a multi-factorial process in which non-scientific arguments tend to play a key role. The aim of this study is to provide biomaterials scientists with information about tracheal research and the animal models used.

Coating with genetic engineered hydrophobin promotes growth of fibroblasts on a hydrophobic solid.

Class I Hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into a highly insoluble amphipathic film. Upon self-assembly of these fungal proteins hydrophobic solids turn hydrophilic, while hydrophilic materials can be made hydrophobic. Hydrophobins thus change the nature of a surface. This property makes them interesting candidates to improve physio- and physico-chemical properties of implant surfaces. We here show that growth of fibroblasts on Teflon can be improved by coating the solid with genetically engineered SC3 hydrophobin. Either deleting a stretch of 25 amino acids at the N-terminus of the mature hydrophobin (TrSC3) or fusing the RGD peptide to this end (RGD-SC3) improved growth of fibroblasts on the solid surface. In addition, we have shown that assembled SC3 and TrSC3 are not toxic when added to the medium of a cell culture of fibroblasts in amounts up to 125 microg ml(-1).

Effect of biologically active coating on biocompatibility of Nitinol devices designed for the closure of intra-atrial communications.

Anti-thrombogenicity and rapid endothelialisation are prerequisites for the use of closure devices of intra-atrial communications in order to reduce the risk of cerebral embolism. The purpose of this study was therefore to assess the effect of bioactive coatings on biocompatibility of Nitinol coils designed for the closure of intra-atrial communications. Nitinol coils (n = 10, each) and flat Nitinol bands (n = 3, each) were treated by basic coating with poly(amino-p-xylylene-co-p-xylylene) and then coated with either heparin, r-hirudin or fibronectin. Anti-thrombogenicity was studied in vitro in a dynamic model with whole blood by partial thromboplastin time (PTT), platelet binding and thrombin generation, respectively, and cytotoxicity by hemolysis. Endothelialisation was studied on Nitinol bands with human umbilical venous endothelial cells (HUVEC) by 3-(4,5-dimethylthiazole-2yl)-2,5-triphenyl tetrazolium (MTT) assay and immnuofluorescence analysis of Ki67, vinculin, fibronectin and von Willebrand Factor. Uncoated or coated devices did not influence hemolysis and PTT. r-Hirudin (but not heparin) and fibronectin coating showed lower platelet binding than uncoated Nitinol (p < 0.005, respectively). Heparin and r-hirudin coating reduced thrombin formation (p < 0.05 versus Nitinol, respectively). HUVEC adhesion, proliferation, and matrix formation decreased in the order: fibronectin coating > uncoated Nitinol > r-hirudin coating > heparin coating > basic coating. MTT assay corroborated these findings. In conclusion, r-hirudin and fibronectin coating, by causing no acute cytotoxicity, decreasing thrombogenicity and increasing endothelialisation improve in vitro biocompatibility of Nitinol devices designed for the closure of intra-atrial communications.

Western blotting as a method for studying cell-biomaterial interactions: the role of protein collection.

Research of cell-biomaterial interactions is building on knowledge and methods available in cell and molecular biology. Western blotting is one of the options to characterize protein expression in cell populations. Method transfer to biomaterial model systems is not trivial because of the structure that exists in many biomaterials, preventing the collection of cell lysate by mechanical means. In this technical report, we describe the influence of different protein collection methods in a model system for cell-biomaterial interactions, consisting of endothelial cells exposed to different stimuli. In particular, the influence of trypsinization before lysis, and handling complexity were determined. The results of this study indicate that many changes in proteins occur because of the intermediate enzymatic treatment, despite the use of ice-cold solutions and protease and tyrosine phosphatase inhibitors throughout the procedure. Protein degradation and slight depressions in molecular weight were observed. The enzymatic treatment induced a changed cell status associated with detachment from the substratum. Western blotting of lysates of cells obtained through enzymatic harvest therefore can only be used with internal controls for the assessment of artifacts introduced by trypsinization, or alternative methods should be sought.

Cell-cycle control in cell-biomaterial interactions: expression of p53 and Ki67 in human umbilical vein endothelial cells in direct contact and extract testing of biomaterials.

Current biocompatibility testing involves the demonstration of cell proliferation, which is usually interpreted as a sign of positive biocompatibility when the materials sustain cell proliferation. As the field of biomaterials research is rapidly moving toward tissue-engineered devices and hybrid organs, control of cell function has become a main topic. Cell function, which involves specific differentiation pathways, cannot be separated from cell-cycle control. The study of cell-cycle control is an important extension of routine proliferation assays and has extensive roots in developmental and tumor biology. We studied the expression of the tumour suppressor gene p53 and the proliferation-associated antigen Ki67 of endothelial cells in response to biomaterial contact. Cells were seeded in six- or 24-well plates, in which one or three 12-mm-diameter biomaterial disks were laid down. After 48- and 72-h incubation periods, cells were processed for flow cytometry, immunofluorescence, or Western blotting. The following materials were used: titanium, NiCr alloy, and CoCr alloy. Cells were also exposed to 24-h (ISO-norm) extracts in 25-cm(2) culture flasks (600, 000 cells) for 24 and 48 h. For extract testing, serially diluted Ni-ion suspensions were also used. Human umbilical vein endothelial cells adhered to metal surfaces and started forming a monolayer within 3 days. Ki67 expression was positive in more than 60% after 2 days and decreased markedly after 3 days of adhesion. During this time cells developed focal contacts and produced a fibronectin matrix. p53 expression could be demonstrated with Western blotting and flow cytometry, but not with immunofluorescence. Differences due to both culturing time and material were found in expression patterns with both methods. Inverse correlations between Ki67 and p53 expression were detected, which are probably based on culture kinetics. The results indicate that expression of p53 and also Ki67 is clearly influenced by biomaterials in direct contact testing, despite the absence of obvious morphological differences. The p53 marker can be used for defining cell function in more detail, although the correlation with specific physiological function has still to be clarified.

A new quantitative test method for cell proliferation based on detection of the Ki-67 protein.

A quantitative method to assess cell proliferation is one essential prerequisite for testing biomaterial cytocompatibility in vitro. Currently used methods, e.g. bromodeoxyuridine incorporation, show serious disadvantages concerning either sensitivity, specificity or handling. A new enzyme linked immunosorbent assay (ELISA) system for the quantification of cell proliferation based on detection of the Ki-67 protein is described. This protein has turned out to be strictly correlated with the active parts of the cell cycle but to be absent in G0. The measurement of Ki-67 expression by different human cell types, e.g. endothelial cells and HeLa cells, was evaluated in order to answer the question of whether the data obtained using the Ki-67 ELISA method correlate with the proliferation measured with flow cytometrical DNA analysis and microscopical evaluation. Methods currently used for the evaluation of cell proliferation were compared to the new Ki-67 ELISA method. In addition, the functionality of adherent endothelial cells, and the viability and morphology of the cells were investigated. Cells were treated with standard culture medium with or without the transcription inhibitor, actinomycin D, or growth factors, e.g. endothelial cell growth factor (ECGF), and were exposed to metal ion standard solutions. These solutions were in a cytotoxic-non-cytotoxic range. Ki-67 ELISA was found to be a reliable quantitative method to assess proliferation of cultured human cells in vitro. It has advantages over methods that are currently being used. It is easy to perform and corresponds to the requirements for a test to be selected for biomaterial testing according to ISO standard 10 993.

Cell adhesion to textured silicone surfaces: the influence of time of adhesion and texture on focal contact and fibronectin fibril formation.

Cell adhesion and spreading on biomaterials is a key issue in the study of cell-biomaterial interactions. With the development of new disciplines within biomaterials research such as tissue engineering and cellular therapy, information at molecular and structural levels is needed in order to conceive and design biomaterials that elicit specific, functional cell responses. In this study we determined the formation of focal adhesions and fibronectin fibrillar structures by human fibroblasts and human umbilical vein endothelial cells adhered to fibronectin-precoated, smooth, and textured silicones as a function of time. Textures consisted of parallel ridges and 0.5 mm deep grooves with a width of 2, 5, and 10 mm. In addition, pillar and well constructs were used. Cells assembled focal adhesions within the first 24 h of adhesion. Fibronectin production and assembly resulted in a dense fibrillar network at day 6. Initial focal adhesion density and size were dictated by the presence of the texture. Topography also influenced initial fibronectin deposition, although the differences did not result in apparent differences in fibronectin networks after 6 days of incubation. Without fibronectin preadsorption, cells did not proliferate on the silicone surfaces. Cells adhered to glass removed all the preabsorbed fibronectin, whereas on silicone they did not. The present study shows that different textures initially give rise to differences in focal contact and fibronectin fibril assembly. The effects of the small, initial in vitro differences on in vivo tissue biocompatibility remains to be studied.