Mesenchymal stem cell adhesion and spreading on nanostructured biomaterials

Bone marrow-derived human mesenchymal stem cells were seeded in serum-free media onto ion beam-deposited nanostructured metalloceramic (Ti-Cr-N) films and plasma-nitrided titanium disks, which were left uncoated as well as precoated with fetal bovine serum. Precoating the disks with serum appears to stimulate cell spreading on both the titanium nitride and metalloceramic materials for as little as 1 hour incubation time. The implication is that both of these materials can adsorb serum proteins in amounts sufficient to influence cell adhesion and spreading for potentially improved in vivo response of orthopedic and dental implants. The materials in this study may prove to exhibit enhanced biological and mechanical properties when compared to conventional micron-scale implant materials such as titanium or cobalt-chrome alloys.     

Shear assay measurements of cell adhesion on biomaterials surfaces

The paper examines the adhesion of human osterosarcoma (HOS) cells to selected biomaterials surfaces that are relevant to implantable biomedical systems and bio-micro-electro-mechanical systems (BioMEMS). The four biomaterials that were explored include: silicon, silicon coated with a nanoscale layer of titanium, Ti–6Al–4V, and poly-di-methy-siloxane (PDMS). The interfacial strengths between the HOS cells and the biomaterials surfaces were determined using a shear assay technique. The adhesion forces were determined using a combination of confocal microscopy images of the three-dimensional cell structure, and computational fluid dynamics (CFD) simulations that coupled actual cell morphologies and non-Newtonian fluid properties in the computation of the adhesion forces. After cell detachment by the shear assay, immunofluorescence staining of the biomedical surfaces was used to reveal the proteins associated with cell detachment. These revealed that the nano-scale Ti coating increases the cell/surface adhesion strength. Silicon with Ti coating has the strongest adhesion strength, while the other surfaces had similar adhesion strength. The measured strengths are shown to be largely associated with the detachment of focal adhesion proteins from extra-cellular matrix (ECM) proteins.     

Multilayer coatings on biomaterials for control of MG-63 osteoblast adhesion and growth

Here, the layer-by-layer technique (LbL) was used to modify glass as model biomaterial with multilayers of chitosan and heparin to control the interaction with MG-63 osteoblast-like cells. Different pH values during multilayer formation were applied to control their physico-chemical properties. In the absence of adhesive proteins like plasma fibronectin (pFN) both plain layers were rather cytophobic. Hence, the preadsorption of pFN was used to enhance cell adhesion which was strongly dependent on pH. Comparing the adhesion promoting effects of pFN with an engineered repeat of the FN III fragment and collagen I which both lack a heparin binding domain it was found that multilayers could bind pFN specifically because only this protein was capable of promoting cell adhesion. Multilayer surfaces that inhibited MG-63 adhesion did also cause a decreased cell growth in the presence of serum, while an enhanced adhesion of cells was connected to an improved cell growth.     

The effect of substrate topography on hFOB cell behavior and initial cell adhesion evaluated by a cytodetacher

This study examined human fetal osteoblast (hFOB) cell morphology, adhesion force, and proliferation on a titanium-coated grooved surface. V-shaped grooves with a depth of 2.4 μm (T1) or 4.8 μm (T2) were produced in silicon wafers using photolithography and wet etching techniques. The grooved substrates were coated with a 200-nm-thick layer of titanium using a sputtering system. Smooth Ti-coated Si wafers were used as control surfaces. Analysis of the scanning electron microscopy observations shows that the cells responded to the micropattern by spreading out and becoming elongated. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay indicated that the grooved specimens had a significantly larger number of cells than did the control group after 5- and 15-day cultures. The cytocompatibility of specimens was quantitatively evaluated by a cytodetacher, which directly measures the detachment shear force of an individual cell to the substrate. After 30-min culture, the cell adhesion forces were 48.4, 136.6, and 103.3 nN for the smooth specimen, the T1 specimen, and the T2 specimen, respectively. The cell adhesion strengths were 294, 501, and 590 Pa for the smooth specimen, the T1 specimen, and the T2 specimen, respectively. The cell adhesion force and cell adhesion strength indicate the quality of cell adhesion, explaining the largest number of cells on grooved specimens. The experimental results suggest that the grooved patterns affect the cell shape and cytoskeletal structure, and thus influence the cell proliferation and cell adhesion force. The cytodetachment test with nanonewton resolution is a sensitive method for studying cell–biomaterial interaction.     

Plasma- and chemical-induced graft polymerization on the surface of starch-based biomaterials aimed at improving cell adhesion and proliferation

Plasma and chemical induced graft polymerization of acrylic monomers on starch-based biomaterials has been performed with the aim to improve cell adhesion and proliferation on the surface of the polymers, in order to adequate their properties for bone tissue engineering scaffolding applications. Plasma and chemical surface activation was aimed to induce the polymerization of acrylic polar monomers being carried out by applying a radio frequency plasma and expose the samples to a mixture of Ar/O₂, or by immersion in a H₂O₂/(NH₄)₂S₂O₈ solution with UV radiation, respectively. Both procedures were followed by the graft polymerization of the corresponding monomers. Polymer grafting was analyzed by Fourier transformed infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) and by contact angle measurements. Properties such as mechanical performance, swelling degree, and degradation behavior, as well as bioactivity, have been studied and compared for the different activation methods. Finally, preliminary cell adhesion and proliferation tests were performed, using goat bone marrow cells, showing a remarkable improvement with respect to original non-surface modified starch-based biomaterials.     

Preliminary study on human protein adsorption and leukocyte adhesion to starch-based biomaterials

In this study, the adsorption of human serum albumin (HSA), fibronectin (FN) and vitronectin (VN) onto the surface of novel biodegradable materials was evaluated by immunostaining. Specifically, polymeric blends of corn starch with cellulose acetate (SCA), ethylene vinyl alcohol copolymer (SEVA-C), and polycaprolactone (SPCL) were immersed in unitary and competitive systems; that is, binary and more complex protein solutions. For binary solutions, different HSA and FN protein distribution patterns were observed depending on the starch-based surface. Furthermore, the relative amount of proteins adsorbed onto starch-based surfaces was clearly affected by protein type: a preferential adsorption of VN and FN as compared to HSA was observed. On tests carried out with unitary, binary and more complex solutions, it was found that vitronectin adsorption ability was enhanced in competitive systems, which was associated with a lower amount of adsorbed albumin. In order to assess the effect of these human proteins on cell behavior, a mixed population of human lymphocytes and monocytes/macrophages was cultured over pre-coated SEVA-C surfaces. Through anti-CD3 and CD-14 monoclonal antibody labeling and cell counting, leukocyte adhesion onto pre-coated SEVA-C surfaces was analyzed. Based on the results, it was possible to detect albumin long-term effects and fibronectin short-term effects on cell adhesion proving that previously adsorbed proteins modulate leukocyte behavior.     

Methylcellulose cell culture as a new cytotoxicity test system for biomaterials

The cytotoxicity of biomaterials can be testedin vitro using various culture systems. Liquid culture systems may detect cytotoxicity of a material either by culture of cells with extracts or with the material itself. In the latter instance, renewing the medium will remove possible released cytotoxic products. The agar-overlay test is a short term semi-solid culture system in which the possible cytotoxicity of biomaterials is identified only by the presence of cell free zones. The aim of this study was to develop a more sensitive cytotoxicity test system for biomaterials, using methylcellulose as a culture gel, mixed with human fibroblasts. The main advantage of the test system is the possibility of evaluating cytotoxicity for a period of up to seven days without renewal of the culture gel. Furthermore it is possible to both quantitatively evaluate by counting absolute cell numbers and to qualitatively evaluate by studying cell morphology with light- and/or electron microscopy. Processed dermal sheep collagen was selected as test material, since contradictory results concerning the cytotoxicity of its extracts have been reported by others [2, 15, 18, 19]. Using our test system, both primary and secondary cytotoxic effects were found. Primary cytotoxicity is due to direct leakage of products from the material, detected by testing, extracts of the collagen or the collagen itself. Secondary cytotoxicity is due to release of cytotoxic products resulting from cell-biomaterial interactions. We conclude that our test system is extremely useful to test materials which are suspected of primary and/or secondary cytotoxicity, either with slow release of cytotoxic products or release of products with late cytotoxic effects.     

Endothelial cell death on biomaterials: Theoretical and practical aspects of investigation

The use of endothelial cell seeding has been proposed as a solution to increase the patency of blood-contacting devices. While there has been a great deal of research into both biological and mechanical mechanisms of failure, relatively little work involving the effects which biomaterials have on the function of cells seeded on such devices has been conducted. We hypothesize, based on previously published results, that endothelial cells attached to synthetic biomaterials exhibit increased intracellular levels of superoxide and other reactive oxygen species. Previous reports have linked elevated levels of reactive oxygen species to NF-κB activation and subsequent cell death via anoikis, a form of apoptosis. Because of this, it is critical that an understanding of how biomaterials affect cellular behavior is developed, and that this knowledge is used in the creation of future devices which rely on cell seeding and cellular ingrowth.     

Injectable percutaneous bone biomaterials: an experimental study in a rabbit model

New percutaneous filling techniques are beginning to be used in bone tumour pathology, because they are less aggressive than surgery. The purpose of this study was to test percutaneous injectable bone biomaterials with a reproducible model. A closed cancellous bone defect was created in the distal femoral extremities of 34 rabbits. Filling was done by a percutaneous injection made on the medial side, after the defect had been hollowed by a lateral approach. Three situations were tested: unfilled, filled with orthopaedic cement, filled with a soft collagen-hydroxyapatite material. Three time intervals (2,4 and 8 weeks) were tested for the three situations. For the mixed hydroxyapatite-collagen material, each component was tested separately: the injectable collagen, and the hydroxyapatite powder. The quality of the defect was assessed by the variations of the defect area on sagittal sections. Bone formation in the defect was quantified for each group and time interval. A bone defect of reproducible size was obtained. Evolution of bone formation was different in each group. An unfilled defect was never completely filled by bone and the defect bone formation rate stayed between 9.9% and 15.1% without any statistical difference between the time intervals. The percutaneous injection of orthopaedic cement, which was progressive, was less complete than an opened surgical filling and explained a frequent low peripheral bone formation. A lower bone formation rate was observed in all the filled groups (orthopaedic cement, hydroxyapatite-collagen, hydroxyapatite and collagen) than in the control unfilled group. This study showed the use of bone biomaterials by an injectable percutaneous method, and a model to evaluate these materials is proposed.     

Influence of nanoporesize on platelet adhesion and activation

In this study we have evaluated the influence of biomaterial nano-topography on platelet adhesion and activation. Nano-porous alumina membranes with pore diameters of 20 and 200 nm were incubated with whole blood and platelet rich plasma. Platelet number, adhesion and activation were determined by using a coulter hematology analyzer, scanning electron microscopy, immunocytochemical staining in combination with light microscopy and by enzyme immunoassay. Special attention was paid to cell morphology, microparticle generation, P-selectin expression and beta-TG production. Very few platelets were found on the 200 nm alumina as compared to the 20 nm membrane. The platelets found on the 20 nm membrane showed signs of activation such as spread morphology and protruding filipodia as well as P-selectin expression. However no microparticles were detected on this surface. Despite the fact that very few platelets were found on the 200 nm alumina in contrast to the 20 nm membrane many microparticles were detected on this surface. Interestingly, all microparticles were found inside circular shaped areas of approximately 3 mum in diameter. Since this is the approximate size of a platelet we speculate that this is evidence of transient, non-adherent platelet contact with the surface, which has triggered platelet microparticle generation. To the authors knowledge, this is the first study that demonstrates how nanotexture can influence platelet microparticle generation. The study highlights the importance of understanding molecular and cellular events on nano-level when designing new biomaterials.     

The effect of starch-based biomaterials on leukocyte adhesion and activation <Emphasis Type="Italic">in vitro</Emphasis>

Leukocyte adhesion to biomaterials has long been recognised as a key element to determine their inflammatory potential. Results regarding leukocyte adhesion and activation are contradictory in some aspects of the material's effect in determining these events. It is clear that together with the wettability or hydrophilicity/hydrophobicity, the roughness of a substrate has a major effect on leukocyte adhesion. Both the chemical and physical properties of a material influence the adsorbed proteins layer which in turn determines the adhesion of cells. In this work polymorphonuclear (PMN) cells and a mixed population of monocytes/macrophages and lymphocytes (mononuclear cells) were cultured separately with a range of starch-based materials and composites with hydroxyapatite (HA). A combination of both reflected light microscopy and scanning electron microscopy (SEM) was used in order to study the leukocyte morphology. The quantification of the enzyme lactate dehydrogenase (LDH) was used to determine the number of viable cells adhered to the polymers. Cell adhesion and activation was characterised by immunocytochemistry based on the expression of several adhesion molecules, crucial in the progress of an inflammatory response. This work supports previous in vitro studies with PMN and monocytes/macrophages, which demonstrated that there are several properties of the materials that can influence and determine their biological response. From our study, monocytes/macrophages and lymphocytes adhere in similar amounts to more hydrophobic (SPCL) and to moderately hydrophilic (SEVA-C) surfaces and do not preferentially adhere to rougher substrates (SCA). Contrarily, more hydrophilic surfaces (SCA) induced higher PMN adhesion and lower activation. In addition, the hydroxyapatite reinforcement induces changes in cell behaviour for some materials but not for others. The observed response to starch-based biodegradable polymers was not significantly different from the control materials. Thus, the results reported herein indicate the low potential of the starch-based biodegradable polymers to induce inflammation especially the HA reinforced composite materials.     

In vivo tests of a novel wound dressing based on biomaterials with tissue adhesion controlled through external stimuli

The high incidence of wounds by second intention and the high costs associated with their treatment give rise to the need for the development of wound dressings that protect not only the wounds themselves but that are also able to promote cell proliferation and skin regeneration. Moreover, it is also very important that no damage to the new regenerated tissue is generated while removing the dressing. In this work, a novel wound dressing, which would be able to favor tissue repair and be removed at an appropriate scheduled moment by means of an external stimulus without promoting extensive damage to the new tissue, was produced and tested. Polyurethane membranes were modified by grafting polymers based on poly(n-isopropylacrylamide) (P-N-IPAAm). P-N-IPAAm undergoes a phase transition at approximately 32°C, which changes its behavior from hydrophilic (below 32°C) to hydrophobic. It was hypothesized that, by reducing the temperature near the wound dressing to values lower than 32°C, the detachment of the dressing would become more effective. The wound dressings containing P-N-IPAAm grafts were tested in vivo by covering excisional wounds produced in mice. The produced dressings were placed in direct contact with the lesions for 3 days. Results showed that the hypothermia due to anesthesia required to remove the dressings from mice lowered the local temperature to 28°C and favored the detachment of the wound dressings containing P-N-IPAAm grafts. Histological analyses showed that lesions covered by dressings presented less intense inflammatory events and denser connective tissue than did the wounds without dressings. The wounds covered by polyurethane membranes with P-N-IPAAm grafts showed signs of more intense re-epithelization and angiogenesis than did the lesions covered by polyurethane without grafts.     

Effects of biomaterials for Lab-on-a-chip production on cell growth and expression of differentiated functions of leukemic cell lines

The rapid increase of the applications for Lab-on-a-chip devices has attracted the interest of researchers and engineers on standard process of the electronics industry for low production costs and large scale development, necessary for disposable applications. The printed circuit board technology could be used for this purpose, in particular for the wide range of materials available. In this paper, assays on biocompatibility of materials used for Lab-on-a-chip fabrication has been carried out using two tumor cell lines growing in suspension, the human chronic myelogenous leukemia K562 cell line, able to undergo erythroid differentiation when cultured with chemical inducers, and the lymphoblastoid cell line (LCL), extensively used for screening of cytotoxic T-lymphocytes (CTLs). We have demonstrated that some materials strongly inhibit cell proliferation of both the two cell lines to an extent higher that 70–75%, but only after a prolonged exposure of 3–6 days (Copper, Gold over Nickel, Aramid fiber filled epoxy uncured, b-stage epoxy die attach film, Tesa 4985 adhesive tape, Pyralux uncured, Copper + 1-octodecanethiol). However, when experiments were performed with short incubation time (1 h), only Aramid fiber filled epoxy uncured was cytotoxic. Variation of the results concerning the other materials was appreciable when the experiments performed on two cell lines were compared together. Furthermore, the effects of the materials on erythroid differentiation and CTL-mediated LCL lysis confirmed, in most of the cases, the data obtained in cytotoxic and antiproliferative tests.     

Effects of adsorbed heat labile serum proteins and fibrinogen on adhesion and apoptosis of monocytes/macrophages on biomaterials

A previously established human monocyte culture protocol was used to determine the effects of varying adsorbed proteins on monocyte/macrophage adhesion and survival on dimethyl-silane (DM) or RGD modified glass coverslips. Cells were allowed to adhere for 2 h in the absence of protein or in the presence of serum, fibrinogen (Fg), heat inactivated serum (HIS), serum supplemented with Fg or HIS with Fg. Cell adhesion and apoptosis rates were determined on days 0 (2 h), 3, 7 and 10 of culture. The presence of serum alone in the initial culture was sufficient to optimize monocyte/macrophage adhesion and survival rates. Adding Fg to serum did not increase adhesion nor decrease apoptotic rates. No protein or the addition of HIS during the initial incubation period significantly decreased monocyte/macrophage adhesion and survival on both surfaces, however, the addition of Fg to HIS restored adhesion and survival rates to those seen with in the presence of serum alone on RGD surfaces. These studies demonstrate that monocyte/macrophage adhesion and survival on biomaterial surfaces are optimized by adsorbed heat labile serum proteins while adsorbed Fg plays a surface property-dependent role.     

Osteoblast cell adhesion on a laser modified zirconia based bioceramic

Due to their attractive mechanical properties, bioinert zirconia bioceramics are frequently used in the high load-bearing sites such as orthopaedic and dental implants, but they are chemically inert and do not naturally form a direct bond with bone and thus do not provide osseointegration. A CO₂ laser was used to modify the surface properties with the aim to achieve osseointegration between bioinert zirconia and bone. The surface characterisation revealed that the surface roughness decreased and solidified microstructure occurred after laser treatment. Higher wettability characteristics generated by the CO₂ laser treatment was primarily due to the enhancement of the surface energy, particularly the polar component, determined by microstructural changes. An in vitro test using human fetal osteoblast cells (hFOB) revealed that osteoblast cells adhere better on the laser treated sample than the untreated sample. The change in the wettability characteristics could be the main mechanism governing the osteoblast cell adhesion on the YPSZ.     

Influence of mineral on the load deformation behavior of polymer in hydroxyapatite-polyacrylic acid nanocomposite biomaterials: a steered molecular dynamics study

Composites of hydroxyapatite and polymers are widely studied for bone replacement. To perform satisfactorily in the human body, these composites need to be biocompatible and exhibit optimum mechanical properties. The load-deformation behavior of composites is often investigated using experimental techniques. However, the molecular mechanisms of load deformation behavior are not clearly understood. We have used Steered Molecular Dynamics to evaluate the load-deformation behavior at interfaces in polyacrylic acid-hydroxyapatite (HAP) composite models. The polymer is pulled at constant velocity in close proximity of HAP. On comparing the results obtained for deformation behavior of polymer in vicinity of mineral and in the absence of mineral, it was found that energy required to pull the polymer in close proximity of HAP is significantly higher. Also, structural details of the load transfer mechanisms in composite were investigated under both conditions. Our simulations indicate that there is a significant role of mineral-polymer interactions on the mechanical response of polymer.     

Improving cell adhesion: development of a biosensor for cell behaviour monitoring by surface grafting of sulfonic groups onto a thermoplastic polyurethane

The surface properties of a material in combination with the mechanical properties are responsible for the material performance in a biological environment as well as the behaviour of the cells which contact with the material. Surface properties such as chemical, physical, biological play an important role in the biomaterials filed. In this work, the surface of a thermoplastic polyurethane film (Elastollan^®1180A50) was tailored with sulfonic groups by grafting [2-(methacryloxyl)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide (SB) after a previous surface activation either by Argon plasma or by ultra-violet irradiation. This surface modification had the purpose of improving cell adhesion in order to develop a biosensor able to monitor cell behaviour. The surfaces were characterized by X-ray photoelectron spectroscopy, by atomic force microscopy and by contact angle measurements in order to evaluate the efficiency of the modification. Additionally, blood compatibility studies and cell adhesion tests with human bone marrow cells were performed. These methods allowed the grafting of SB and the results indicate that a higher density of grafting was achieved with previous surface plasma treatment than with UV irradiation. However, for both techniques, the presence of SB functional groups led to a decrease of hydrophobicity and roughness of the surface, together with an improvement of the materials biological performance.     

Dynamic blood cell contact with biomaterials: validation of a flow chamber system according to international standards

The increasing number of patients requiring prosthetic substitution of segments of the vascular system strongly supports the need to optimize a relevant, standardized testing panel for new materials designed for synthetic vascular prostheses. The ISO gives the standard requirements for testing biomaterials provided for implantation. Our primary interest was the establishment of a reliable in vitro panel as a useful and relevant screening system for vascular implant devices to evaluate blood/device interactions under flow conditions. The aim of the present study was to evaluate influences of different flow conditions on blood cell–biomaterial interactions with special emphasis on the interactions of human granulocytes (PMN) and polymeric surfaces. PMN were isolated and vital cells were quantified by flow cytometrical analysis directly before, as well as immediately after the experiments. The viscosity of the final cellular suspension was analysed by using a computerized cone-plate rheometer. As reference materials we used FEP-teflon, PVC-DEHD, PU, PP and PE. Dacron and ePTFE synthetic vascular protheses were tested in a comparative way to those references. The adhesion processes were observed over a period of 40 minutes under arterial (shear stress 0.74 Pa) and venous (shear stress 0.16 Pa) flow conditions in a parallel plate flow chamber system under highly standardized conditions and laminar flow. The cells were observed with the help of inverse light microscopy. Cell behaviour was recorded and analysed in both analogue (video) and digital (imaging system) modes. Samples of the cell suspensions were obtained at regular time intervals and analysed by enzyme linked immuno sorbent assay (ELISA) to quantify LTB4 release. Irrespective of the material, approximately 3 to 4 times more PMN adhered to the biomaterial surfaces under venous flow conditions compared to the arterial. Shear intensity did not influence the running order of biomaterials with respect to cell numbers. This response in descending order at the end of the experiments was as follows: PU, PVC-DEHD, PP, PE and ePTFE. The biochemical analyses indicate that in the system used only a weak effect on LTB4 release induced by the different materials could be determined. A significant effect caused by flow conditions was not observed. Further experiments, both static as well as dynamic, must be performed for multiple, relevant parameters of haemocompatibility, for potential biomaterials as well as those currently in use in vascular prostheses.