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Macrophage behavior upon biomaterial implantation conditions the inflammatory response and subsequent tissue repair. The hypothesis behind this work was that fibrinogen (Fg) and magnesium (Mg) biomaterials, used in combination (FgMg) could act synergistically to modulate macrophage activation, promoting a pro-regenerative phenotype. Materials were characterized by scanning electron microscopy, Fg and Mg degradation products were quantified by atomic absorption spectroscopy and ELISA. Whole blood immune cells and primary human monocyte-derived macrophages were exposed to the biomaterials extracts in unstimulated (M0) or pro-inflammatory LPS or LPS-IFNγ (M1) conditions. Macrophage phenotype was evaluated by flow cytometry, cytokines secreted by whole blood cells and macrophages were measured by ELISA, and signaling pathways were probed by Western blotting. The secretomes of macrophages preconditioned with biomaterials extracts were incubated with human mesenchymal stem/stromal cells (MSC) and their effect on osteogenic differentiation was evaluated via Alkaline Phosphatase (ALP) activity and alizarin red staining. Scaffolds of Fg, alone or in the FgMg combination, presented similar 3D porous architectures. Extracts from FgMg materials reduced LPS-induced TNF-α secretion by innate immune cells, and macrophage M1 polarization upon LPS-IFNγ stimulation, resulting in lower cell surface CD86 expression, lower NFκB p65 phosphorylation and reduced TNF-α secretion. Moreover, while biomaterial extracts per se did not enhance MSC osteogenic differentiation, macrophage secretome, particularly from cells exposed to FgMg extracts, increased MSC ALP activity and alizarin red staining, compared with extracts alone. These findings suggest that the combination of Fg and Mg synergistically influences macrophage pro-inflammatory activation and crosstalk with MSC. STATEMENT OF SIGNIFICANCE: Modulating macrophage phenotype by degradable and bioactive biomaterials is an increasingly explored strategy to promote tissue repair/regeneration. Fibrinogen (Fg) and magnesium (Mg)-based materials have been explored in this context. Previous work from our group showed that monocytes interact with fibrinogen adsorbed onto chitosan surfaces through TLR4 and that fibrinogen scaffolds promote in vivo bone regeneration. Also, magnesium ions have been reported to modulate macrophage pro-inflammatory M1 stimulation and to promote bone repair. Here we report, for the first time, the combination of Fg and Mg materials, hypothesizing that it could act synergistically on macrophages, directing them towards a pro-regenerative phenotype. As a first step towards proving/disproving our hypothesis we used extracts obtained from Fg, Mg and FgMg multilayer constructs. We observed that FgMg extracts led to a reduction in the polarization of macrophages towards a pro-inflammatory phenotype. Also, the secretome of macrophages exposed to extracts of the combination material promoted the expression of osteogenic markers by MSCs., Competing Interests: Declaration of Competing Interest There are no conflicts to declare., (Copyright © 2020. Published by Elsevier Ltd.)

Biomedical devices in the blood flow disturb the fine-tuned balance of pro- and anti-coagulant factors in blood and vessel wall. Numerous technologies have been suggested to reduce coagulant and inflammatory responses of the body towards the device material, ranging from camouflage effects to permanent activity and further to a responsive interaction with the host systems. However, not all types of modification are suitable for all types of medical products. This review has a focus on application-oriented considerations of hemocompatible surface fittings. Thus, passive versus bioactive modifications are discussed along with the control of protein adsorption, stability of the immobilization, and the type of bioactive substance, biological or synthetic. Further considerations are related to the target system, whether enzymes or cells should be addressed in arterial or venous system, or whether the blood vessel wall is addressed. Recent developments like feedback controlled or self-renewing systems for drug release or addressing cellular regulation pathways of blood platelets and endothelial cells are paradigms for a generation of blood contacting devices, which are hemocompatible by cooperation with the host system. STATEMENT OF SIGNIFICANCE: This paper is part 4 of a series of 4 reviews discussing the problem of biomaterial associated thrombogenicity. The objective was to highlight features of broad agreement and provide commentary on those aspects of the problem that were subject to dispute. We hope that future investigators will update these reviews as new scholarship resolves the uncertainties of today., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The development of new biomaterials to be used in tissue engineering applications is creating new solutions for a range of healthcare problems. The trend in biomaterials research has shifted from biocompatible "immune-evasive" biomaterials to "immune-interactive" materials that modulate the inflammatory response supporting implant integration as well as improving healing and tissue regeneration. Inflammasomes are large intracellular multiprotein complexes that are key players in host defence during innate immune responses and assemble after recognition of pathogens or danger signals. The process of biomaterial implantation causes injury to tissues that will consequently release danger signals that could be sensed by the inflammasome. There are increasing evidences that the inflammasome has a role in several inflammatory processes, from pathogen clearance to chronic inflammation or tissue repair. Thus, modulation of the inflammasome activity appears as an important target in the development of effective approaches in regenerative medicine. In this review, we discuss the main points of the current understanding on the host response to implanted biomaterials and how the paradigm of "immune-evasive" biomaterials has shifted over the last years; the significance of the inflammasome in the inflammatory response to biomaterials; and the growing idea that the immune system is of key importance in an effective tissue repair and regeneration. STATEMENT OF SIGNIFICANCE: We herein discuss the main points of the current understanding on the host response to implanted biomaterials and how the paradigm of "immune-evasive" biomaterials has shifted to "immune-interactive" over the last years; the significance of the inflammasome in the inflammatory response to biomaterials; and the growing idea that the immune system is of key importance in an effective tissue repair and regeneration, supporting the emerging concept of Regenerative Immunology. The inflammasome is a recent and central concept in immunology research. Since the beginning of this century the inflammasome is viewed as key platform of the innate immune response. We believe that, successful modulation of the inflammasome activity will become a milestone in the fields of tissue engineering and regenerative medicine., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The biological response to implanted biomaterials is a complex and highly coordinated phenomenon involving many different cell types that interact within 3D microenvironments. Here, we increased the complexity of a 3D platform to include at least 3 cell types that play a role in the host response upon scaffold implantation. With this system, it was possible to address how immune responses triggered by 3D biomaterials mediate recruitment of stromal cells that promote tissue regeneration, mesenchymal stromal/stem cells (MSC), or a foreign body response, fibroblasts. Primary human macrophages yielded the highest fibroblast recruitment when interacting with chitosan scaffolds but not polylactic acid. Interestingly, when there were MSC and fibroblasts in the same environment, macrophages in chitosan scaffolds again promoted a significant increase on fibroblast recruitment, but not of MSC. However, macrophages that were firstly allowed to interact with MSC within the scaffolds were no longer able to recruit fibroblasts. This study illustrates the potential to use different scaffolds to regulate the dynamics of recruitment of proregenerative or fibrotic cell types through immunomodulation. Overall, this work strengths the idea that ex vivo predictive systems need to consider the different players involved in the biological response to biomaterials and that timing of arrival of specific cell types will affect the outcome., (Copyright © 2017 John Wiley & Sons, Ltd.)

In our search for immunomodulatory biomaterials capable of modulating the inflammatory response through M2 macrophage polarization, we report here on a new strategy that resulted from the incorporation of resolvin D1 (RvD1), a pro-resolution lipid mediator in porous 3D chitosan (Ch) scaffolds, followed by its lyophilisation. We have investigated the inflammatory response caused by this biomaterial in vivo using a mouse air-pouch model of inflammation. We found that this developed material caused a decrease in inflammatory cells recruited to the implant site, together with higher numbers of F4/80(+)/CD206(+) cells (M2 macrophages) and lower numbers of F4/80(+)/CCR7(+) cells (M1 macrophages). It also induced a general decrease in pro-inflammatory cytokines, and caused a decrease in the inflammatory cells observed around and within the implanted scaffolds, when compared with Ch alone or Ch not submitted to lyophilisation after RvD1 incorporation. Our results demonstrate that we were able to develop an immunomodulating biomaterial that triggers a shift in the macrophage response towards a M2 reparative response that will be advantageous for the host., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Collagen has become a key-molecule in cell culture studies and in the tissue engineering field. Industrially, the principal sources of collagen are calf skin and bones which, however, could be associated to risks of serious disease transmission. In fact, collagen derived from alternative and riskless sources is required, and marine organisms are among the safest and recently exploited ones. Sea urchins possess a circular area of soft tissue surrounding the mouth, the peristomial membrane (PM), mainly composed by mammalian-like collagen. The PM of the edible sea urchin Paracentrotus lividus therefore represents a potential unexploited collagen source, easily obtainable as a food industry waste product. Our results demonstrate that it is possible to extract native collagen fibrils from the PM and produce suitable substrates for in vitro system. The obtained matrices appear as a homogeneous fibrillar network (mean fibril diameter 30-400 nm and mesh < 2 μm) and display remarkable mechanical properties in term of stiffness (146 ± 48 MPa) and viscosity (60.98 ± 52.07 GPa·s). In vitro tests with horse pbMSC show a good biocompatibility in terms of overall cell growth. The obtained results indicate that the sea urchin P. lividus can be a valuable low-cost collagen source for mechanically resistant biomedical devices.

Designing new biomaterials that can modulate the inflammatory response instead of attempting just to reduce it constitutes a paradigm change in regenerative medicine. This work aimed to investigate the capacity of an immunomodulatory biomaterial to enhance bone regeneration. For that purpose we incorporated a molecule with well-established pro-inflammatory and pro-healing roles, fibrinogen, in chitosan scaffolds. Two different incorporation strategies were tested, leading to concentrations of 0.54±0.10mg fibrinogen g(-1) scaffold immediately upon adsorption (Fg-Sol), and 0.34±0.04mg fibrinogen g(-1) scaffold after washing (Fg-Ads). These materials were implanted in a critical size bone defect in rats. At two months post-implantation the extent of bone regeneration was examined by histology and the systemic immune response triggered was evaluated by determining the percentages of myeloid cells, T and B lymphocytes in the draining lymph nodes. The results obtained indicate that the fibrinogen incorporation strategy conditioned the osteogenic capacity of biomaterials. Fg-Ads scaffolds led to more bone formation, and the presence of Fg stimulated angiogenesis. Furthermore, animals implanted with Fg-Ads scaffolds showed significant increases in the percentages of B lymphocytes and myeloid cells in the draining lymph nodes, while levels of T lymphocytes were not significantly different. Finally, a significant increase in TGF-β1 was detected in the plasma of animals implanted with Fg-Ads. Taken together the results presented suggest a potential correlation between the elicited immune response and biomaterial osteogenic performance., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

In this study, (125)I-radiolabelling was explored to follow the kinetics and isotherm of fibronectin (FN) adsorption to porous polymeric scaffolds, as well as to assess the elution and exchangeability of pre-adsorbed FN following incubation in serum-containing culture medium. Chitosan (CH) porous scaffolds with two different degrees of acetylation (DA 4% and 15%) were incubated in FN solutions with concentrations ranging from 5 to 50 µg/mL. The kinetic and isotherm of FN adsorption to CH were successfully followed using (125)I-FN as a tracer molecule. While on DA 4% the levels of adsorbed FN increased linearly with FN solution concentration, on DA 15% a saturation plateau was attained, and FN adsorbed amounts were significantly lower. These findings were supported by immunofluorescent studies that revealed, for the same FN solution concentration, higher levels of exposed cell-binding domains on DA 4% as compared with DA 15%. Following incubation in serum containing medium, DA 4% also revealed higher ability to exchange pre-adsorbed FN by new FN molecules from serum than DA 15%. In accordance, when assessing the efficacy of passively adsorbed FN to promote endothelial cell (EC) adhesion to CH, ECs were found to adhere at higher levels to DA 4% as compared with DA 15%, 5 µg/mL of FN being already efficient in promoting cell adhesion and cytoskeletal organization on CH with DA 4%. Taken together the results show that protein radiolabelling can be used as an effective tool to study protein adsorption to porous polymeric scaffolds, both from single and complex protein solutions.

Albumin-scavenging surfaces decorated with n-alkyl chains represent an established strategy for blood-contacting applications. To evaluate this concept, a set of poly (2-hydroxyethyl methacrylate) (pHEMA) films modified with different amounts of octadecyl isocyanate (C18) was investigated in an in vitro hemocompatibility assay using freshly drawn human whole blood. In addition, the hydrogel materials were thoroughly characterized with respect to changes in wettability and elasticity, which accompanied the gradual chemical modification of pHEMA. An increase of the surface C18 content induced enhanced hydrophobicity and stiffness. Immobilization of C18 chains was found to substantially reduce the coagulation activation and the complement activation by the pHEMA films. Platelet adhesion and degranulation (PF4 release) were similar on the modified and the unmodified pHEMA. Platelet adhesion to pHEMA hydrogels was lower than the polytetrafluoroethylene reference. We conclude that the immobilization of octadecyl chains improved the hemocompatibility of pHEMA materials under conditions that might be encountered in low shear blood flow., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Thrombus formation, due to thrombin generation, is a major problem affecting blood-contacting medical devices. This work aimed to develop a new strategy to improve the hemocompatibility of such devices by the immobilization of a naturally occurring thrombin inhibitor into a nanostructured surface. Boophilin, a direct thrombin inhibitor from the cattle tick Rhipicephalus microplus, was produced as a recombinant protein in Pichia pastoris. Boophilin was biotinylated and immobilized on biotin-terminated self-assembled monolayers (SAM) via neutravidin. In order to maintain its proteinase inhibitory capacity after surface immobilization, boophilin was biotinylated after the formation of a boophilin-thrombin complex to minimize the biotinylation of the residues involved in thrombin-boophilin interaction. The extent of boophilin biotinylation was determined using matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. Boophilin immobilization and thrombin adsorption were quantified using quartz crystal microbalance with dissipation. Thrombin competitive adsorption from human serum was assessed using ¹²⁵I-thrombin. Thrombin inhibition and plasma clotting time were determined using spectrophotometric techniques. Boophilin-coated SAM were able to promote thrombin adsorption in a selective way, inhibiting most of its activity and delaying plasma coagulation in comparison with boophilin-free surfaces, demonstrating boophilin's potential to improve the hemocompatibility of biomaterials used in the production of blood-contacting devices., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Macrophages and dendritic cells (DC) share the same precursor and play key roles in immunity. Modulation of their behaviour to achieve an optimal host response towards an implanted device is still a challenge. Here we compare the differentiation process and polarisation of these related cell populations and show that they exhibit different responses to chitosan (Ch), with human monocyte-derived macrophages polarising towards an anti-inflammatory phenotype while their DC counterparts display pro-inflammatory features. Macrophages and DC, whose interactions with biomaterials are frequently analysed using fully differentiated cells, were cultured directly on Ch films, rather than exposed to the polymer after complete differentiation. Ch was the sole stimulating factor and activated both macrophages and DC, without leading to significant T cell proliferation. After 10 d on Ch, macrophages significantly down-regulated expression of pro-inflammatory markers, CD86 and MHCII. Production of pro-inflammatory cytokines, particularly TNF-α, decreased with time for cells cultured on Ch, while anti-inflammatory IL-10 and TGF-β1, significantly increased. Altogether, these results suggest an M2c polarisation. Also, macrophage matrix metalloproteinase activity was augmented and cell motility was stimulated by Ch. Conversely, DC significantly enhanced CD86 expression, reduced IL-10 secretion and increased TNF-α and IL-1β levels. Our findings indicate that cells with a common precursor may display different responses, when challenged by the same biomaterial. Moreover, they help to further comprehend macrophage/DC interactions with Ch and the balance between pro- and anti-inflammatory signals associated with implant biomaterials. We propose that an overall pro-inflammatory reaction may hide the expression of anti-inflammatory cytokines, likely relevant for tissue repair/regeneration.

The remarkable properties of poly-aminoacids, mainly their biocompatibility and biodegradability, have prompted an increasing interest in these polymers for biomedical applications. Poly-γ-glutamic acid (γ-PGA) is one of the most interesting poly-aminoacids with potential applications as a biomaterial. Here we describe the production and characterization of γ-PGA by Bacillus subtilis natto. The γ-PGA was produced with low molecular weight (10-50 kDa), high purity grade (>99 %) and a D: -/L: -glutamate ratio of 50-60/50-40 %. To evaluate the feasibility of using this γ-PGA as a biomaterial, chitosan (Ch)/γ-PGA nanoparticles were prepared by the coacervation method at pH ranging from 3.0 to 5.0, with dimensions in the interval 214-221 nm with a poly-dispersion index of ca. 0.2. The high purity of γ-PGA produced by this method, which is firstly described here, renders this biopolymer suitable for biomedical applications. Moreover, the Ch/γ-PGA nanocomplexes developed in this investigation can be combined with biologically active substances for their delivery in the organism. The fact that the assembly between Ch and γ-PGA relies on electrostatic interactions enables addition of other molecules that can be released into the medium through changes from acidic to physiological pH, without loss in biological activity.

An exacerbated inflammatory response questions biomaterial biocompatibility, but on the other hand, inflammation has a central role in the regulation of tissue regeneration. Therefore, it may be argued that an 'ideal' inflammatory response is crucial to achieve efficient tissue repair/regeneration. Natural killer (NK) cells, being one of the first populations arriving at an injury site, can have an important role in regulating bone repair/regeneration, particularly through interactions with mesenchymal stem/stromal cells (MSCs). Here, we studied how biomaterials designed to incorporate inflammatory signals affected NK cell behaviour and NK cell-MSC interactions. Adsorption of the pro-inflammatory molecule fibrinogen (Fg) to chitosan films led to a 1.5-fold increase in adhesion of peripheral blood human NK cells, without an increase in cytokine secretion. Most importantly, it was found that NK cells are capable of stimulating a threefold increase in human bone marrow MSC invasion, a key event taking place in tissue repair, but did not affect the expression of the differentiation marker alkaline phosphatase (ALP). Of significant importance, this NK cell-mediated MSC recruitment was modulated by Fg adsorption. Designing novel biomaterials leading to rational modulation of the inflammatory response is proposed as an alternative to current bone regeneration strategies.

Last trends in Biomaterials focus the mimic of cellular environments capable to control cellular responses. Epidermal growth factor (EGF) is a pleiotropic cytokine known to regulate cell proliferation, differentiation, and death. This study aims to optimize the immobilization of EGF on 11-mercapto-1-undecyl-tetra(ethylene)glycol (EG4)-self-assembled monolayers (SAMs) and to establish a new model surface to study EGF-mediated signaling. Gold substrates were modified with a monolayer of EG4 and N,N'-carbonyldiimidazole (CDI) was used to activate hydroxyl terminated groups of EG4-SAMs. EGF was then immobilized on activated EG4-SAMs at pH 7.4, 4 degrees C, and 100 rpm. Different immobilization reaction times were tested as well as different CDI concentrations to optimize the reaction conditions and obtain a range of immobilized EGF concentrations on the surfaces. Surface characterization of EGF-SAMs was performed using radiolabeling, water contact angle measurements, X-ray photoelectron spectroscopy, and ELISA. Phosphorylation of EGFR on BT-20 breast cancer cell line by EGF-SAMs was tested by immunostaining. EGF was successfully immobilized on EG4-SAMs, at 4 degrees C and pH 7.4 in a range of concentrations from 3.6 +/- 0.8 to 17.6 +/- 1.5 ng/cm(2). The concentration of EGF increases with immobilization time and with the CDI concentration reaching the maximum for surfaces activated with 30 mg/mL of CDI after 48 h. The bioactivity of EGF-SAMs was confirmed by immunostaining of phospho-EGFR of BT-20 cells. This study described EGF immobilization on EG4-SAMs at different concentrations, which could be important surface models to study specific protein interactions at the molecular level evolving EGF-family of proteins., ((c) 2010 Wiley Periodicals, Inc.)

A polymeric solution and a reinforcement phase can work as an injectable material to fill up bone defects. However, the properties of the solution should be suitable to enable the transport of that extra phase. Additionally, the use of biocompatible materials is a requirement for tissue regeneration. Thus, we intended to optimize a biocompatible polymeric solution able to carry hydroxyapatite microspheres into bone defects using an orthopedic injectable device. To achieve that goal, polymers usually regarded as biocompatible were selected, namely sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and Na-alginate (ALG). The rheological properties of the polymeric solutions at different concentrations were assessed by viscosimetry before and after moist heat sterilization. In order to correlate rheological properties with injectability, solutions were tested using an orthopedic device applied for minimal invasive surgeries. Among the three polymers, ALG solutions presented the most suitable properties for our goal and a non-sterile ALG 6% solution was successfully used to perform preliminary injection tests of hydroxyapatite microspheres. Sterile ALG 7.25% solution was found to closely match non-sterile ALG 6% properties and it was selected as the optimal vehicle. Finally, sterile ALG 7.25% physical stability was studied at different temperatures over a 3-month period. It was observed that its rheological properties presented minor changes when stored at 25 degrees C or at 4 degrees C.

Chitosan was functionalized with imidazole moieties (CHimi) with the aim of improving its buffering capacity and promoting the endosomal escape ability of chitosan-DNA complexes, ultimately increasing their transfection efficiency. 5.6%, 12.9% and 22.1% of the glucosamine residues of chitosan were substituted. Complexes with different molar ratios of primary amines to DNA phosphate anion (N/P) were prepared by a coacervation method. For an N/P>3, CHimi polymers are able to complex electrostatically with DNA and condense it into positively charged nanostructures (average size 260 nm and zeta potential +16 mV at pH 5.5). In the concentration range 2.5-100 microg ml(-1), the modified polymers had no cytotoxic effect on 293T cells. CHimi polymers with the highest degree of substitution were found to enhance beta-gal expression in 293T and HepG2 cells. Bafilomycin A1 inhibited transfection, indicating that the protonation of the imidazole groups in the endolysosome pathway favors the escape of the complexes from the endosomes, increasing the amount of transgene that can reach the cell nucleus.

The objective of this study was to compare the biological effects of two key cell-adhesive proteins, fibronectin (FN) and vitronectin (VN), upon adsorption onto molecularly-designed model surfaces. Single-component and mixed self-assembled monolayers (SAMs) of alkanethiols on gold with OH and CH(3) terminal groups were prepared at 100%, 65%, 36% and 0% of OH at the surface, to generate a range of surfaces with a simple chemistry and a wettability gradient. FN and VN were adsorbed under non-competitive (single-protein solutions) and competitive (multi-protein solutions) conditions, and compared at different levels: adsorbed amount (radiolabelling), elution, functional presentation of cell-binding domains (ELISA), and role in mediating cell adhesion (antibody-based assay). The observed trends were related to mesenchymal stem cell response in terms of adhesion and overall cell morphology. Under non-competitive conditions, adsorption of both proteins increased with surface hydrophobicity. The presence of competitive proteins significantly decreased the adsorbed amounts, although both proteins were still detected in all SAMs. Adsorption of FN followed a trend similar to that of non-competitive conditions, while adsorption of VN was higher on 100%OH-SAMs. Concerning elution, retention of adsorbed VN was always higher than that of FN. For both proteins, functional presentation of cell-binding domains was more effective on the more hydrophilic 100%OH-SAMs. This fact, coupled to the ability of this type of SAMs to selectively recruit and retain VN in the presence of competitive serum proteins, seems to correlate with the better cell response observed on these surfaces, as compared with hydrophobic 0%OH(100%CH(3))-SAMs.

The effect of surface wettability on fibrinogen adsorption, platelet adhesion and platelet activation was investigated using self-assembled monolayers (SAMs) containing different ratios of longer chain methyl- and shorter chain hydroxyl-terminated alkanethiols (C15CH3 vs. C11OH) on gold. Protein adsorption studies were performed using radiolabeled human fibrinogen (HFG). Platelet adhesion and activation studies with and without pre-adsorbed fibrinogen, albumin and plasma were assessed using scanning electron microscopy (SEM) and a glutaraldehyde-induced fluorescence technique (GIFT). Results demonstrated a linear decrease of HFG adsorption with the increase of OH groups on the monolayer (increase of the hydrophilicity). Platelet adhesion and activation also decrease with increase of hydrophilicity of surface. Concerning SAMs pre-immersed in proteins, fibrinogen adsorption was related with high platelet adhesion and activation. The passivant effect of albumin on platelet adhesion and activation was only demonstrated on SAMs contained C11OH. When all the blood proteins are present (plasma) platelet adhesion was almost absent on SAMs with 65% and 100% C11OH. This could be explained by the higher albumin affinity of the SAMs with 65% C11OH and the lower total protein adsorption associated with SAMs with 100% C11OH.

In the present work, a novel route for the preparation of porous ceramic microspheres is described. Two ceramic powders, calcium-titanium-phosphate (CTP) and hydroxyapatite (HAp), were mixed with a sodium alginate solution that enabled the preparation of spherical particles, using the droplet extrusion method combined with ionotropic gelation in the presence of Ca(2+). The spherical particles were subsequently sintered, to burn-off the polymer and obtain calcium-phosphate microspheres with a uniform size and an interconnected porous network. CTP microspheres with diameters ranging from 513 +/- 24 microm to 792 +/- 35 microm and with pores of approximately 40 microm were obtained. HAp microspheres presented diameters of 429 +/- 46 microm and 632 +/- 40 microm and pores of ca. 2 microm. Depending on the formulations tested, the structure of both calcium phosphates may become altered during the sintering process, suggesting that the ratio between the ceramic phase and the polymer solution is a critical parameter. Porous microspheres prepared using the described methodology are promising candidates as bone defect fillers and scaffolds for bone tissue regeneration.

The contribution of the surface chemistry of an implant to the thickness of the fibrous capsule formed after implantation was herein investigated. For that, self-assembled monolayers (SAMs) of alkanethiols on gold with different terminal functional groups (COOH, OH, and CH(3)) were used. These surfaces were implanted in subcutaneous air pouches of BALB/c mice and the ensuing fibrous capsules were evaluated and compared with the initial inflammatory response caused by the implant. The thickness of the fibrous capsules that are under organization around the implant was measured 1 week after implantation by histology. Inflammatory exudates were collected from the air pouches 24 h after the implantation of SAMs and were analyzed by flow cytometry. A significant increase in the thickness of fibrous capsules was seen around implanted CH(3)-terminated SAMs, and also in gold surfaces, in comparison with the air pouch wall of sham-operated mice and of COOH- and OH-covered SAMs. The CH(3)-coated implants also recruited higher numbers of inflammatory cells; this enhancement involved a significant number of Mac-1(+) cells. Our data indicate that implant surfaces coated with CH(3) induce thick fibrous capsules and this may be the result of the stronger inflammatory effect of CH(3) in comparison with COOH or OH chemical groups.

In order to understand the effect of titanium ions on the molecular structure of hydroxyapatite (HAp), HAp powders were incubated in solutions with different titanium concentrations. After incubation, the powders obtained were analysed using different techniques, namely X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), differential thermal analysis (DTA), X-ray photoelectron spectroscopy (XPS), and energy dispersive spectroscopy (EDS). The results suggest that, depending on the concentration of titanium in solution, two different mechanisms of interaction with HAp occur. For concentrations equal to or smaller than 200 ppm, the titanium uptake by the solid seems to be primarily due to incorporation in the lattice. For higher concentrations, a dissolution-precipitation process seems to occur, leading to formation of a titanium phosphate compound.

We have used self-assembled monolayers (SAMs) of alkanethiolates on gold to study the contribution of methyl terminal functional groups in implant-triggered inflammation. The CH3-coated biomaterials were inserted in an air-pouch cavity of the BALB/c mouse and the in situ inflammatory response was monitored 4, 24, 48 and 72 h later. Flow cytometry was applied to define surface expression of the adhesion receptor Mac-1 (CD11b/CD18), a marker of activated leukocytes, and also of CD3 and B220 antigens (T and B lymphocytes). The CH3-coated surfaces caused a significant enhancement in the number of Mac-1+ cells in the implant. The only significant change in T and B lymphocytes was a transient increase in T cells detected 48 h after the implantation. Peak numbers of Mac-1+ phagocytes were observed 24 h after implantation. We conclude that if CH3 is present at the surface of implants, this chemical group will trigger a significant enhancement of activated phagocytes involved in the inflammatory reaction, and this phenomenon may extend the local phlogistic event.

The biocompatibility of chemoenzymatically generated dextran-acrylate hydrogels has been evaluated in vitro, using human foreskin fibroblasts, and in vivo, by subcutaneous and intramuscular implantation in Wistar rats for up to 40 days. In vitro tests show that hydrogel extracts only minimally reduced (<10%) the mitochondrial metabolic activity of fibroblasts. Direct contact of the hydrogels with cells induced a cellular proliferation inhibition index (CPII) of 50-80%, compared with a control, whereas through indirect contact, the CPII values were <16%, suggesting that the high CPII values achieved in the direct assay test were likely due to mechanical stress or limitations in oxygen diffusion. Hence, the hydrogels were noncytotoxic. Moreover, cell-material interaction studies show that these hydrogels were nonadhesive. Finally, histologic evaluation of tissue response to subcutaneous and intramuscular implants showed acceptable levels of biocompatibility, as characterized by a normal cellular response and the absence of necrosis of the surrounding tissues of the implant. In the first 10 days, the foreign-body reaction in the intramuscular implantation was more severe than in subcutaneous implantation, becoming identical after 30 days. In both cases, dextran hydrogels did not show signs of degradation 6 weeks postimplantation and were surrounded by a thin fibrous capsule and some macrophages and giant cells. This response is typical with a number of nondegradable biocompatible materials. These results indicate that dextran hydrogels are biocompatible, and may have suitable applications as implantable long-term peptide/protein delivery systems or scaffolds for tissue engineering., (Copyright 2004 Wiley Periodicals, Inc.)

The effect of surface composition and wettability on the adsorption of human serum albumin (HSA) was studied. Self-assembled monolayers (SAMs) containing mixtures of longer chain methyl- and shorter chain hydroxyl-terminated alkanethiols on gold were used to produce a range of surfaces with different wettabilities and exposed functional groups. Different SAMs were characterized by X-ray photoelectron spectroscopy, water contact angles, and Fourier transform infrared reflection absorption spectroscopy (IRAS). HSA adsorption onto the different SAMs was evaluated by contact angle measurements (wetting tension determinations), radiolabeling of proteins, and IRAS. Concerning HSA adsorption, all the techniques demonstrated higher HSA adsorption on more hydrophobic surfaces. The wetting tension measurements and IRAS suggested a gradual decrease of the HSA adsorption with increases of surface hydrophilicity. Radiolabeled albumin measurements also demonstrated a significant decrease of HSA adsorption on the pure hydroxyl-terminated SAMs. However, no significant differences were detected between mixed and pure methyl-terminated SAMs. Studies of HSA exchangeability with human fibrinogen have suggested that an ideal percentage of hydroxyl groups on the surface may increase albumin affinity without fibrinogen adsorption., (Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 158-171, 2003)

The adhesion of human leukocytes to self-assembled monolayers of well-defined surface chemistry was investigated in vitro. Polymorphonuclear (PMN) and mononuclear leukocytes were isolated from human blood by centrifugation techniques. The effect on adhesion of cell activation produced by pre-incubation of leukocytes with phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) was also studied. Gold substrates were modified by treatment with alkanethiols with three different terminal chemical groups: COOH, OH, and CH(3). After incubation with the two subpopulations of leukocytes, the monolayers were washed, treated with fixative, stained with a Giemsa method, and observed by light microscopy to quantify the number of attached leukocytes. Comparative quantification of the density of leukocyte adhesion to the three types of self-assembled monolayers was determined. The hydrophobic surface expressing CH(3) was found to be the one that induced the highest adhesion density of leukocytes, both of PMN and mononuclear cells. In vitro activation of both mononuclear and PMN leukocytes further increased cell adhesion to the chemically defined monolayers that were used. This enhancement was higher for PHA-activated than for PMA-stimulated mononuclear cells, whereas PMA treatment of neutrophils resulted in a higher rate of adhesion of these cells than PHA stimulation., (Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 65A: 429-434, 2003)

Femoral implantation of regenerated cellulose hydrogels revealed their biocompatibility, but a complete osseointegration could not be observed. Phosphorylation was therefore envisaged as the means to enhance cellulose bioactivity. In vitro studies showed that regenerated cellulose hydrogels promote bone cells attachment and proliferation but do not mineralize in acellular simulated physiological conditions. On the contrary, phosphorylated cellulose has shown an opposite behavior, by inducing the formation of a calcium phosphate layer in simulated physiological conditions, but behaving as a poor substrate for bone cells attachment and proliferation. In order to investigate the in vivo behavior of these materials, and assess the influence of mineralization induction ability vs. bone cells compatibility, unmodified and phosphorylated cellulose hydrogels were implanted in rabbits for a maximum period of 6 months and bone regeneration was investigated. Despite the difficulties arising from the retraction of cellulose hydrogels upon dehydration during the preparation of retrieved implants, histological observations showed no inflammatory response after implantation, with bone intra-spongious regeneration of cells and the integration of the unmodified as well as the phosphorylated cellulose implants. After a maximum implantation period of 6 months, histological observations, histomorphometry and the measurement of the amount of 45Ca incorporated in the surrounding tissue indicated a slightly better osseointegration of phosphorylated cellulose, although no significant differences between the two materials were found.

An ideal surface for many biomedical applications would resist non-specific protein adsorption while at the same time triggering a specific biological pathway. Based on the approach of selectively binding albumin to free fatty acids, stearyl groups were immobilized onto poly(styrene) backbone via poly(ethylene oxide) side chains. X-ray photoelectron spectroscopy (XPS) analysis indicates substantial surface enrichment of the stearyl poly(ethylene oxide) (SPEO). In an aqueous environment, the surface rearrangement is limited, as proved by dynamic contact angle tests. The comb-like copolymer presents a special hydrophobic surface with high SPEO surface density, which may be due to the 'tail like' SPEO architecture at the copolymer/water interface. Protein adsorption tests confirm that the comb-like surfaces adsorb high levels of albumin and resist fibrinogen adsorption very significantly. The surfaces prepared in this research attract and reversibly bind albumin due to the synergistic action of the PEO chains and the stearyl end groups.

The formation of an apatite-like layer was achieved by immersing Ti-6A1-4V, Ti-Al-2.5Fe, and 316 L stainless-steel substrata in Hank's balanced salt solution (HBSS). The layer was characterized by surface analysis techniques, namely X-ray microanalysis and X-ray diffraction, and the morphology was observed by scanning electron microscopy and atomic force microscopy. The concentrations of Ca and P were monitored as a function of time. The morphology of the precipitate layer seems to be dependent both on the type of metal substrate and its surface finish. Polished Ti-6A1-4V and Ti-Al-2.5Fe surfaces exhibit a plate precipitate morphology, whereas rougher surfaces show scattered crystal-like precipitation. The results suggest that the layer produced by immersion of polished titanium alloys in HBSS is constituted by an amorphous apatite.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) analyses have been used to investigate the dissolution of hydroxyapatite in the presence of titanium chloride suggesting the substitution of titanium ions for calcium in the hydroxyapatite structure. The surface analytical data suggest that titanium was incorporated within the solid phase as titanium phosphate. Comparison of relative ion intensities in the ToF-SIMS spectra reveals changes in the composition of the surface chemistry of hydroxyapatite. These relative ion intensities show that the maximum surface uptake of titanium occurred at a solution concentration of 500 ppm titanium chloride for a time of incubation of 30 min. These data correlate with that obtained using XPS highlighting the semi-quantitative information which ToF-SIMS can provide.

The electrochemical dissolution behaviour of Ti6Al4V alloy coated with hydroxyapatite (HA) by plasma spraying was studied in Hank's balanced salt solution (HBSS) and compared with that of polished and grit-blasted passivated surfaces. Two different nominal thicknesses of HA (50 and 200 micro m) were used. Taking a polished passivated surface as reference, grit blasting of the substrate increased the electrical charge used in the oxidation of Ti6Al4V alloy at constant potential, as a result of increased surface area. However, only HA coatings with a thickness of 200 micro m were capable of reducing the charge to values lower than those measured for polished surfaces. Electrochemical impedance spectroscopy has also shown that only 200 micro m thick coatings are effective in reducing the oxidation rate of the substrate. Furthermore, in potentiostatic experiments the 50 micro m thick coating detached from the substrate, which did not occur with the 200 micro m thick coating. However, after 6 months immersion in HBSS, detachment occurred in some regions of both coatings. No titanium, aluminium or vanadium were detected in solution by electrothermal atomic absorption spectroscopy. These data indicate that HA is an effective barrier to metal ion release, even for the thinner coatings, due to formation of metal phosphates or to incorporation of metal ions in the HA structure.

In vitro studies to determine Fe3+ levels in mice liver samples were performed using platinum microelectrodes and atomic absorption spectrophotometry. Microelectrodes have been shown to be useful for quantitative analysis of metal ions released during the biodegradation process that occurs on implanted metallic biomaterials.