Segmented poly(esterurethane urea)s from novel urea-diol chain extenders: synthesis, characterization and in vitro biological properties.

This work describes the preparation, physicochemical characterization, mechanical properties and in vitro biological properties of two bioresorbable aliphatic segmented poly(esterurethane urea)s (SPEUU) based on poly(epsilon-caprolactone) diol (PCL diol), 1,6-hexamethylene diisocyanate and two novel urea-diol chain extenders. To strengthen the interactions through hydrogen bonding in the hard segments of SPEUU, novel chain extenders containing urea groups were synthesized and used in the SPEUU formulation. The different chemical structures of the chain extenders modulated the phase separation of soft and hard segments, as demonstrated by the thermal behavior. The hard segment association was enhanced using a diurea-diol chain extender. The biological interactions between the obtained materials and blood were studied by in vitro methods. Research on the protein adsorption, platelet adhesion and thrombus formation is presented. Studies of protein adsorption onto polymeric surfaces showed that SPEUU adsorbed more albumin than fibrinogen. Studies on platelet adhesion and thrombus formation of SPEUU-coated coverslips indicated the antithrombogenic behavior of these surfaces. The synthesized SPEUU revealed no signs of cytotoxicity to Chinese hamster ovary cells, showing satisfactory cytocompatibility.

Synthesis and characterization of membranes obtained by graft copolymerization of 2-hydroxyethyl methacrylate and acrylic acid onto chitosan.

Chitosan based membranes to be applied on wound healing as topical drug delivery systems were developed by graft copolymerization of acrylic acid (AA) and 2-hydroxyethyl methacrylate (HEMA) onto chitosan using cerium ammonium nitrate as chemical initiator. Evidence for graft copolymerization of the vinyl monomers onto chitosan was obtained by FTIR and DMTA. Swelling degree, cytotoxicity, thrombogenicity and haemolytic activity of these membranes were evaluated. Chitosan-graft-AA-graft-HEMA showed to be the best matrix for drug delivery systems than chitosan-graft-AA because it retains good swelling properties, but the content in HEMA has improved cytocompatibility, hemocompatibility and thrombogenic character.

Preparation of polymeric urease discs by an electron beam irradiation technique.

A preparation method of immobilized urease discs by an electron beam irradiation technique was developed, and the relationship between enzyme activity and preparation conditions was investigated. The immobilized urease disc was a thin circular film (200 microm, 5 mm phi) that is useful for biomedical applications. The activity of urease irradiated with 1 Mrad at room temperature was protected by the presence of cysteine. The activity of the immobilized urease discs was studied as a function of monomer concentration (80-90%) and the thicker disc gave a high activity. The durability of the immobilized urease discs gave a high activity. The durability of the immobilized urease discs was evaluated by repeated batch enzyme reactions, and a high activity yield (80-85%) was obtained.

Alpha-tricalcium phosphate cement: "in vitro" cytotoxicity.

Calcium phosphate-based bioceramics have revolutionized orthopedic and dental repair of damaged parts of the bone system. Among these materials, calcium phosphate-based cements, with hydraulic setting, stand out due to their biocompatibility and in situ hardening, which allow easy manipulation and adaptation to the shape and dimensions of bone defects. An investigation was made of the in vitro cytotoxic effect of calcium phosphate cement based on alpha-tricalcium phosphate, immersed for different lengths of time in simulated body fluid (SBF), based on the ISO-10993 "Biological Evaluation of Medical Devices" standard. The culture medium was Chinese hamster ovary (CHO) cells in contact with diluted cement extracts. The results revealed that the calcium phosphate cement used was cytotoxic and that the material's cytotoxicity decreased the longer the cement was immersed in SBF.

Preparation of high-quality iodine-125-labelled pituitary luteinizing hormone for radioimmunoassay.

High quality pituitary luteinizing hormone labelled with 125-I was obtained after separating out the more heavily iodinated fractions, through starch gel electrophoresis, using the cathodal component (Fraction 1) which was further purified on Sephadex G-100, giving an almost pure 125-I-labelled luteinizing hormone preparation, presenting excellent immunoreactivity and low levels of damage on incubation in plasma. The quality control of the steps of the technique was done with plasma-coated talc (200 mg) which compared favorably, as far as being an indication of intact labelled luteinizing hormone, with the more time-consuming chromatoelectrophoresis.

Cytotoxicity due to corrosion of ear piercing studs.

It is well known that allergic and/or inflammatory reactions can be elicited from the use of gold-coated studs, particularly the type used for piercing ears, since they are left in contact with body fluids until the puncture heals. Inasmuch as gold is known as a non-toxic element, other elements of the substrate material may be responsible for some allergies. Therefore, characteristics of the coating, such as defects that expose the substrate to the human skin or body fluids, play an important role in the development of skin sensitization. In this study, the cytotoxicity of commercial studs used for ear piercing and laboratory-made studs was determined in a culture of mammalian cells. The corrosion performance of the studs was investigated by means of weight loss measurements and electrochemical impedance spectroscopy. The elements that leached out into the medium were also analysed by instrumental neutron activation analysis. Further, the surfaces of the studs were examined by scanning electron microscopy and analysed by energy dispersive spectroscopy to identify defects and reaction products on the surface, both before and after their exposure to the culture medium. The stud which showed lower corrosion performance resulted in higher cytotoxicity. Ti showed no cytotoxicity and high corrosion resistance, proving to be a potential material for the manufacture of ear piercing studs.

Blood compatibility of tubular polymeric materials studied by biological surface interactions.

Tubular polymeric materials modified by radiation techniques can be used as vascular prosthesis and components of prosthetic devices. The biological interaction between these materials and blood was studied by in vitro and ex vivo methods. Silicone rubber tubes were copolymerized with acrylamide and N-vinylpyrrolidone by radiation-grafting techniques. The irradiation was performed with gamma-rays from a 60Co source at a constant dose rate (0.2 kGy/h) for various time intervals (4-15 h). To evaluate the antithrombogenicity of the grafted tubes, the surface adsorption of 125I-albumin and 125I-fibrinogen was studied. All graft copolymers show a preference for albumin, and the degree of preference appears to correlate with antithrombogenic tendency. In the ex vivo experiment with animals, tubes were implanted in the carotid artery of dogs and the blood flow in the graft copolymers was detected with an ultrasonic flow meter. The blood flow rate in the ungrafted implants decreased more rapidly (stopped completely after 15 to 210 min) compared to the flow rate in the grafted ones (decreased slowly from 38 to 35 ml/min and 70 to 60 ml/min). There was a direct relationship between both methods in the study of blood compatibility of the materials. The results suggest that the graft copolymers can be used as biomaterials for long-term use in cardiovascular systems.

The interaction of blood proteins with alpha-alumina.

The use of alumina (alpha-Al2O3) as a material for cardiovascular applications was investigated on the basis of protein adsorption and thrombus formation on the material. The adsorption of 125I-labelled albumin and fibrinogen from phosphate buffered saline (pH 7.35, 0.100 M NaCl, 8.66 mM KH2PO4 and 41 mM Na2HPO4) solution on ceramic discs of alumina was studied. Both albumin and fibrinogen presented affinity for ceramic surfaces, with adsorptions of 1.47 +/- 0.06 ng/cm2 and 0.198 +/- 0.01 ng/cm2, respectively. Scanning electron micrographs of the alpha-Al2O3 surfaces after contact of the discs with whole human blood showed a thrombogenic behavior of alumina alpha. These results indicate a hemoincompatible property. Although critical surface tension (gamma C: 21.8 dynes/cm) of the disc surfaces determined by contact angle technique of sessile drops indicates that alumina alpha is a biocompatible material, by this criterion, the data reported here indicate that alpha-Al2O3 cannot be used for cardiovascular applications.

Nanocomposite hydroxyapatite formation on a Ti-13Nb-13Zr alloy exposed in a MEM cell culture medium and the effect of H2O2 addition.

Titanium alloys are known to nucleate an apatite layer when in contact with simulated body fluid. This improves the bioactivity of titanium implants and accelerates osseointegration. Promoting the formation of hydroxyapatite on biocompatible metals is, therefore, a very important topic of biomaterials research. In this paper, the formation of hydroxyapatite (HA) on the near-beta Ti-13Nb-13Zr alloy by immersion in minimal essential medium (MEM), with and without H(2)O(2) addition, has been studied using electrochemicals methods, scanning electron microscopy and X-ray photoelectron spectroscopy. The in vitro biocompatibility of this alloy was evaluated by cytotoxicity tests. The Ti-13Nb-13Zr alloy exhibits passive behaviour over a wide potential range in MEM and the passive film is composed of an inner barrier layer and an outer porous layer. The addition of H(2)O(2) leads to a thickening of the outer porous layer and strongly reduced current density. With regard to the surface composition, immersion in MEM solution results in the formation of an island-like distribution of HA+amino acids. Addition of H(2)O(2) to the MEM solution strongly promotes the formation of a thicker, continuous but porous nanocomposite layer of HA+amino acids. The Ti-13Nb-13Zr alloy is non-toxic and the nanocomposite HA+amino acid layer formed in the MEM solution favours the growth of osteoblast cells. For Ti alloys, the release of H(2)O(2) in the anti-inflammatory response appears to be an important beneficial process as it accelerates osseointegration.

Production of porous hydroxyapatite by the gel-casting of foams and cytotoxic evaluation.

This study presents the manufacture of highly porous hydroxyapatite by a novel technique that employs the foaming of suspensions prior to the in situ polymerization of organic monomers contained in the compositions. This method produces strong gelled bodies with up to 90% porosity that can withstand machining in the green state. Complex-shaped components can be obtained if the process comprises casting in one of the processing steps. The organic additives are eliminated at temperatures above 300 degrees C, and sintering is carried out for consolidation of the ceramic matrix. Spherical interconnected cells with sizes ranging from 20 to 1000 micrometer characterize the porous structure, depending on the specimen density. Cytotoxicity tests were conducted on extracts from sintered HA foams based on a quantitative method of cell colony formation and the determination of cell death after indirect contact of the porous material with mammalian cells. This in vitro test of biological evaluation revealed that the original purity of the biomedical-grade hydroxyapatite powder was affected neither through processing nor by the employed reagents.

The "so-called" somatomedin B and growth hormone measured by radioimmunoassay in normal and cirrhotic individuals.

Serum somatomedin B (SmB) levels in cirrhotic individuals, 3.3 +/- 1.5 mg/l, were strikingly lower (P less than 0.001) than in normal subjects, 9.0 +/- 1.7 mg/l. SmB levels were clearly related to the levels of alpha 2-globulins in the cirrhotics (r = + 0.8, P less than 0.002). Serum SmB and growth hormone correlated negatively in a group of normal and cirrhotic individuals (r = -0.67, P less than 0.001). Direct measurements of serum SmB failed to reveal differences between hepatic, renal and peripheral veins. These findings suggest that: 1) SmB is produced by liver and/or normal liver function plays an important role in maintaining normal serum SmB levels; 2) SmB carrier proteins are reduced in the cirrhotics and 3) SmB is part of a negative feed-back system involving growth hormone.

Hemocompatible properties of polymeric derivative of paracetamol.

Copolymerization of N,N-dimethylacrylamide (DMAA) and p-acryloyloxiacetanilide (AOA) was carried out at different mole ratios of the monomers to obtain copolymers of varying compositions. DMAA contents were very near to the corresponding monomer feed and varied between 0.20 and 0.80. Investigation of the protein adsorption of these polymer surfaces showed that copolymers with higher DMAA content adsorbed more albumin than fibrinogen. The scanning electron micrographs of the polymer-coated coverslips after contact with blood showed an antithrombogenic behaviour of these surfaces.

Surface studies of albumin immobilized onto PE and PVC films.

The aim of this study is to evaluate the thrombogenic behaviour of the low density polyethylene and poly(vinyl chloride) modified by radiation-grafting technique. After copolymerization with acrylic acid by gamma-rays from a 60Co source, BSA was immobilized onto functionalized graft copolymers. The biological interaction between these materials and blood was studies by in vitro methods. The BSA immobilization effectively suppressed the adhesion and activation of platelets when it contacted whole blood.

Adsorption of plasma proteins to DMAA hydrogels obtained by ionizing radiation and its relationship with blood compatibility.

The interaction of plasma proteins such as albumin, gamma-globulin, and fibrinogen with the surface of graft copolymers DMAA-G-PTFE, DMAA-G-PETFE, and DMAA-G-PE obtained by radiation graft polymerization was studied. The adsorption of serum proteins was affected by the hydrophilicity of the graft copolymers. Increased albumin adsorption and decreased fibrinogen and gamma-globulin adsorption with increasing grafting levels was shown. A certain range of degrees of grafting showed an improved blood compatibility of the polymeric surfaces due to the existence of a hydrophilic/hydrophobic balance on the polymers. The results suggest that the DMAA-G-PTFE, DMAA-G-PETFE, and DMAA-G-PE graft copolymers can be used as biomaterials for long-term use in cardiovascular systems.

Increased levels of multiplication-stimulating activity, an insulin-like growth factor, in fetal rat serum.

Multiplication-stimulating activity (MSA), purified from medium conditioned by the BRL-3A rat liver cell line, previously has been shown to be closely related to the human somatomedins or insulin-like growth factors. A radioimmunoassay was utilized to measure MSA levels in sera from fetal, maternal, and young rats. A serum somatomedin-binding protein was found to interfere in the radioimmunoassay by competing with antibody for binding 125I-labeled MSA. Therefore, prior to radioimmunoassay, sera were filtered on Sephadex G-75 in 1 M acetic acid to dissociate and separate somatomedin activity from the binding protein. Concentrations of MSA by radioimmunoassay were 20- to 100-fold higher in feta rat sera (1.8-4.4 micrograms/ml) than in maternal sera. MSA levels gradually decreased after birth, reaching maternal levels by day 25 of extrauterine life. MSA concentrations in feta rat sera also were found to be correspondingly high by a rat liver membrane radioreceptor assay and a competitive binding protein assay using rat serum somatomedin-binding protein. The findings of higher levels of MSA in fetal than in maternal rat sera and the gradual decline in MSA serum concentrations after birth are in direct contrast to total somatomedin activities measured by biosassay. Thus, MSA may function as a growth factor in the fetal rat whereas other somatomedins may play a role in stimulating growth during extrauterine life.

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.

Blood compatibility of tubular polymeric materials studied by biological surface interactions

Tubular polymeric materials modified by radiation techniques can be used as vascular prosthesis and components of prosthetic devices. The biological interction between these materials and blood was studied by in vitro and ex vivo methods. Silicone rubber tubes were copolymerized with acrylamide and N-vinylpyrrolidone by radiation-grafting techniques. The irradiation was performed with y-rays from a 60Co source at a constant dose rate (0.2 kGy/h) for various time intervals (4-15 h). To evaluate the antithrombogenicity of the grafted tubes, the surface adsorption of 125I-albumin and 125I-fibrinogen was studied. All graft copolymers show a preference for albumin, and the degree of preference appears to correlate with antithrombogenic tendency. In the ex vivo experiment with animals, tubes were implanted in the carotid artery of dogs and the blood flow in the graft copolymers was detected with an ultrasonic flow meter. The blood flow rate in the ungrafted implants decreased more rapidly (stopped completely after 15 to 210 min) compared to the flow rate in the grafted ones (decreased slowly from 38 to 35 ml/min and 70 to 60 ml/min). There was a direct relationship between both methods in the study of blood compatibility of the materials. The results suggest that the graft copolymers can be used as biomaterials for long-term use in cardiovascular systems

The interation of blood proteins with alpha-alumina

The use of alumina (Ó-Al2O3) as a material for cardiovascular applications was investigated on the basis of protein adsorption and thrombus formation on the material. The adsorption of 125I-labelled albumin and fibrinogen from phosphate buffered saline (pH 7.35, 0.100 M NaCl, 8.66 mM KH2PO4 and 41 mM Na2HPO4) solution on ceramic discs of alumina was studied. Both albumin and fibrinogen presented affinity for ceramic surfaces, with adsorptions of 1.47 + or - 0.6 ng/cm2 and 0.198 + or - 0.01 ng/cm2, respectively. Scanning electron micrographs of the Ó-Al2O3 surfaces after contact of the discs with whole human blood showed a thrombogenic behavior of alumina alpha. These results indicate a hemoincompatible property. Although critical surface tension (YC: 21.8 dynes/cm) of the disc surfaces determined by contact angle technique of sessile drops indicates that alumina alpha is a biocompatible material, by this criterion, the data reported here indicate that Ó-Al2O3 cannot be used for cardiovascular applications