Novel antibiotic-loaded orthopaedic bone cements: insights on drug release profiles and biocompatibility

Acrylic bone cement (BC) is widely used as an anchor of artificial joints. Bacterial infection due to biofilm formation and inflammation are common and difficult to treat problems associated with commercial available BC formulations. Research on novel BC compositions is urgently needed. The main objective of this thesis was to develop a new biocompatible antibiotic-loaded BC with improved release profile. To achieve that aim several additives were incorporated, as an antibiotic (levofloxacin) to combat bacterial growth, an anti-inflammatory drug (diclofenac) to decrease the inflammatory process and two well-known and broadly used biopolymers, alginate and chitosan in order to increase matrix porosity, and in this way to intensify the amount of released drug. Novel BC formulations were tested in order to find the most suitable one that had potential to proceed to clinical application. Numerous tests were conducted as: a) evaluation of drug release profiles in different biomimetic media, b) mechanical and surface studies, c) microbiological activity testing against Staphylococcus aureus and d) in vitro biocompatibility assays (fibroblasts and osteoblasts). In general, the addition of biopolymers increased drug release, didn’t compromised BC mechanical properties and increased BC hydrophilicity. Microbiological testing revealed that Lev[BC]Chi was the only matrix that reduced significantly biofilm formation. On the contrary, alginate and diclofenac loading into BC seemed to increase biofilm growth. Biocompatibility studies showed some decrease in cell viability, in particularly on osteoblasts, mainly due to the high amounts of released drugs. In conclusion, the present work has shown that the matrix with more potential to proceed in further investigations was Lev[BC]Chi. Other conditions (namely additives and drugs concentrations) should be evaluated with the other tested BC matrices before being discharged.

1,4-Dioxane enhances properties and biocompatibility of polyanionic collagen for tissue engineering applications

Polyanionic collagen obtained from bovine pericardial tissue submitted to alkaline hydrolysis is an acellular matrix with strong potential in tissue engineering. However, increasing the carboxyl content reduces fibril formation and thermal stability compared to the native tissues. In the present work, we propose a chemical protocol based on the association of alkaline hydrolysis with 1,4-dioxane treatment to either attenuate or revert the drastic structural modifications promoted by alkaline treatments. For the characterization of the polyanionic membranes treated with 1,4-dioxane, we found that (1) scanning electron microscopy (SEM) shows a stronger reorientation and aggregation of collagen microfibrils; (2) histological evaluation reveals recovering of the alignment of collagen fibers and reassociation with elastic fibers; (3) differential scanning calorimetry (DSC) shows an increase in thermal stability; and (4) in biocompatibility assays there is a normal attachment, morphology and proliferation associated with high survival of the mouse fibroblast cell line NIH3T3 in reconstituted membranes, which behave as native membranes. Our conclusions reinforce the ability of 1,4-dioxane to enhance the properties of negatively charged polyanionic collagen associated with its potential use as biomaterials for grafting, cationic drug- or cell-delivery systems and for the coating of cardiovascular devices.

Design and characterization of novel biopolymeric structure using biocompatible ionic liquids

The main objective of this thesis was the development of polymeric structures from the dissolution of FucoPol, a bacterial exopolysaccharide (EPS), in a biocompatible ionic liquid, choline acetate. The FucoPol was produced by the bacteria Enterobacter A47 using glycerol as carbon source at controlled temperature and pH (30ºC and 7, respectively). At the end of 3 days it was produced 7 g/L of FucoPol. The net yield of Fucopol in glycerol (YP/S) was 0.22 g/g and the maximum productivity 2.37 g/L.d This polymer was characterized about its composition in sugars and acyl groups (by High-Performance Liquid Chromatography - HPLC), containing fucose (35 % mol), galactose (21 % mol), glucose (29 % mol), rhamnose (3% mol) and glucuronic acid (12% mol) as well as acetate (14.28 % mol), pyruvate (2.15 % mol) and succinate (1.80 % mol). Its content of water and ash was 15% p/p and 2% p/p, respectively, and the chemical bonds (determined by Infrared Spectroscopy - FT-IR) are consistent to the literature reports. However, due to limitations in Differential Scanning Calorimetry (DSC) equipment it was not possible to determine the glass transition temperature. In turn, the ionic liquid showed the typical behavior of a Newtonian fluid, glass transition temperature (determined by DSC) -98.03ºC and density 1.1031 g/cm3. The study of chemical bonds by FT-IR showed that amount of water (8.80%) influenced the visualization of the bands predicted to in view of their chemical structure. After the dissolution of the FucoPol in the ionic liquid at different temperatures (50, 60, 80 and 100 ° C) it was promoted the removal of this by the phase inversion method using deionized water as a solvent, followed by drying in an oven at 70 ° C. The mixtures before and after the phase inversion method were characterized through the studies mentioned above. In order to explore possible application field’s biocompatibility assays and collage on balsa wood tests were performed. It was found that the process of washing with water by the phase inversion method was not totally effective in removing the biocompatible ionic liquid, since all FucoPol – IL mixtures still contained ionic liquid in their composition as can be seen by the DSC results and FT-IR. In addition, washing the mixtures with water significantly altered the composition of FucoPol. However, these mixtures, that developed a viscous behavior typical of a non-Newtonian fluid (shear-thinning), have the potential to be applied in the biomedical field as well as biological glues.

Avaliação in vitro da citotoxicidade de resinas acrílicas para reembasamento baseadas em uma nova formulação

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Histological evaluation of biocompatibility of lyophilized bovine pericardium implanted subcutaneously in rats

This article aims at investigating in vivo evaluation of lyophilization procedure on the biocompatibility of bovine pericardium treated with glutaraldehyde (GA). The bovine pericardium was fixed with 0.5% glutaraldehyde during 10 days and preserved in 4% formaldehyde (FA). Two groups of samples were prepared from treated membranes: Group 1, nonlyophilized samples and Group 2, lyophilized samples. Male Sprague-Dawley rats (4 weeks after birth) were anesthetized (pentobarbital sodium 25 mg/kg of body weight) and in each one were implanted subcutaneously in the dorsal region a sample from Group 1 and another from Group 2. These samples were explanted after 30 days for histological analysis. No intercurrences took place after the surgery. No differences (P > 0.05) in the calcification, granulomatous reaction, mononuclear infiltration, and granulation tissue development was observed between both groups. The implanted lyophilized samples presented a trend for a reduced inflammatory reaction. Lyophilization of the bovine pericardium does not seem to increase the above listed tissue reaction.

Ex vivo biocompatibility tests of regular and white forms of mineral trioxide aggregate

Aim: To examine the genotoxicity and cytotoxicity of regular and white mineral trioxide aggregate (MTA) ex vivo by the single-cell gel (comet) assay and trypan blue exclusion test, respectively. Methodology: Aliquots of 1 × 10 4 Chinese hamster ovary cells were incubated at 37°C for 3 h with grey and white forms of MTA at final concentrations ranging from 1 to 1000 μg mL -1. The negative control group was treated with vehicle control phosphate buffer solution for 3 h at 37°C and the positive control group was treated with methyl metasulfonate (at 1 μg mL -1) for 1 h at 37°C. After incubation, the cells were centrifuged at 180 g for 5 min and washed twice with fresh medium and resuspended with fresh medium. Each individual treatment was repeated three times consecutively to ensure reproducibility. Parameters from single-cell gel (comet) and cytotoxicity assays were assessed by the Kruskal-Wallis nonparametric test. Results: Neither compounds produced genotoxic effects with respect to the single-cell gel (comet) assay in all concentrations evaluated. In the same way, the dose-response relationships of all compounds tested at concentrations ranging from 1 to 1000 μg mL -1 on cell viability assessed by the trypan blue assay displayed no statistically significant differences (P > 0.05) for either endodontic material. Conclusions: Regular (grey) and white MTA are not genotoxins and do not induce cellular death. © 2006 International Endodontic Journal.

Hancornia speciosa latex for biomedical applications: physical and chemical properties, biocompatibility assessment and angiogenic activity

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Biocompatibility assessment of the first generation PediaFlow pediatric ventricular assist device

The PediaFlow pediatric ventricular assist device is a miniature magnetically levitated mixed flow pump under development for circulatory support of newborns and infants (3-15 kg) with a targeted flow range of 0.3-1.5 L/min. The first generation design of the PediaFlow (PF1) was manufactured with a weight of approximately 100 g, priming volume less than 2 mL, length of 51 mm, outer diameter of 28 mm, and with 5-mm blood ports. PF1 was evaluated in an in vitro flow loop for 6 h and implanted in ovines for three chronic experiments of 6, 17, and 10 days. In the in vitro test, normalized index of hemolysis was 0.0087 ± 0.0024 g/100L. Hemodynamic performance and blood biocompatibility of PF1 were characterized in vivo by measurements of plasma free hemoglobin, plasma fibrinogen, total plasma protein, and with novel flow cytometric assays to quantify circulating activated ovine platelets. The mean plasma free hemoglobin values for the three chronic studies were 4.6 ± 2.7, 13.3 ± 7.9, and 8.8 ± 3.3 mg/dL, respectively. Platelet activation was low for portions of several studies but consistently rose along with observed animal and pump complications. The PF1 prototype generated promising results in terms of low hemolysis and platelet activation in the absence of complications. Hemodynamic results validated the magnetic bearing design and provided the platform for design iterations to meet the objective of providing circulatory support for young children with exceptional biocompatibility.

Enhanced biocompatibility of Nitinol via surface treatment and alloying

It is projected that by 2020, there will be 138 million Americans over 45, the age at which the increased incidence of heart diseases is documented. Many will require stents. This multi-billion dollar industry, with over 2 million patients worldwide, 15% of whom use Nitinol stents have experienced a decline in sales recently, due in part to thrombosis. It is a sudden blood clot that forms inside stents. As a result, the Food and Drug Administration and American Heart Association are calling for a new generation of stents, new designs and different alloys that are more adaptable to the arteries. The future of Nitinol therefore depends on a better understanding of the mechanisms by which Nitinol surfaces can be rendered stable and inert. In this investigation, binary and ternary Nitinol alloys were prepared and subjected to various surface treatments such as electropolishing (EP), magnetoelectropolishing (MEP) and water boiling & passivation (W&P). In vitro corrosion tests were conducted on Nitinol alloys in accordance with ASTM F 2129-08. The metal ions released into the electrolyte during corrosion tests were measured by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). Biocompatibility was assessed by observing the growth of human umbilical vein endothelial cells (HUVEC) on the surface of Nitinol alloys. Static and dynamic immersion tests were performed by immersing the Nitinol alloys in cell culture media and measuring the amount of metal ions released in solution. Sulforhodamine B (SRB) assays were performed to elucidate the effect of metal ions on the growth of HUVEC cells. The surfaces of the alloys were studied using Scanning Electron Microscopy (SEM) and X-ray Photoelectron Spectroscopy (XPS) respectively. Finally, wettability and surface energy were measured by Contact Angle Meter, whereas surface roughness was measured by Atomic Force Microscopy (AFM). All the surface treated alloys exhibited high resistance to corrosion when compared with untreated alloys. SRB assays revealed that Ni and Cu ions exhibited greater toxicity than Cr, Ta and Ti ions on HUVEC cells. EP and MEP alloys possessed relatively smooth surfaces and some were composed of nickel oxides instead of elemental nickel as determined by XPS. MEP exhibited lowest surface energy and lowest surface roughness.

Biocompatibility assessment of biosorbable polymer coated nitinol alloys

Owing to an increased risk of aging population and a higher incidence of coronary artery disease (CAD), there is a need for more reliable and safer treatments. Numerous varieties of durable polymer-coated drug eluting stents (DES) are available in the market in order to mitigate in-stent restenosis. However, there are certain issues regarding their usage such as delayed arterial healing, thrombosis, inflammation, toxic corrosion by-products, mechanical stability and degradation. As a result, significant amount of research has to be devoted to the improvement of biodegradable polymer-coated implant materials in an effort to enhance their bioactive response. ^ In this investigation, magneto-electropolished (MEP) and a novel biodegradable polymer coated ternary Nitinol alloys, NiTiTa and NiTiCr were prepared to study their bio and hemocompatibility properties. The initial interaction of a biomaterial with its surroundings is dependent on its surface characteristics such as, composition, corrosion resistance, work of adhesion and morphology. In-vitro corrosion tests such as potentiodynamic polarization and electrochemical impedance spectroscopic (EIS) studies were conducted to determine the coating stability and longevity. In-vitro hemocompatibility studies and HUVEC cell growth was performed to determine their thrombogenic and biocompatibility properties. Critical delamination load of the polymer coated Nitinol alloys was determined using Nano-scratch analysis. Sulforhodamine B (SRB) assays were performed to elucidate the effect of metal ions leached from Nitinol alloys on the viability of HUVEC cells. Scanning Electron Microscopy (SEM), X-ray Photoelectron Spectroscopy (XPS), contact angle meter and X-ray diffraction (XRD) were used to characterize the surface of the alloys. ^ MEP treated and polymer coated (PC) Nitinol alloys displayed a corrosion resistant polymer coating as compared to uncoated alloys. MEP and PC has resulted in reduced Ni and Cr ion leaching from NiTi5Cr and subsequently low cytotoxicity. Thrombogenicity tests revealed significantly less platelet adhesion and confluent endothelial cell growth on polymer coated and uncoated ternary MEP Nitinol alloys. Finally, this research addresses the bio and hemocompatibility of MEP + PC ternary Nitinol alloys that could be used to manufacture blood contacting devices such as stents and vascular implants which can lead to lower U.S. healthcare spending.^

Enhanced Biocompatibility of NiTi (Nitinol) Via Surface Treatment and Alloying

It is projected that by 2020, there will be 138 million Americans over 45, the age at which the increased incidence of heart diseases is documented. Many will require stents. This multi-billion dollar industry, with over 2 million patients worldwide, 15% of whom use Nitinol stents have experienced a decline in sales recently, due in part to thrombosis. It is a sudden blood clot that forms inside stents. As a result, the Food and Drug Administration and American Heart Association are calling for a new generation of stents, new designs and different alloys that are more adaptable to the arteries. The future of Nitinol therefore depends on a better understanding of the mechanisms by which Nitinol surfaces can be rendered stable and inert. In this investigation, binary and ternary Nitinol alloys were prepared and subjected to various surface treatments such as electropolishing (EP), magnetoelectropolishing (MEP) and water boiling & passivation (W&P). In vitro corrosion tests were conducted on Nitinol alloys in accordance with ASTM F 2129-08. The metal ions released into the electrolyte during corrosion tests were measured by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). Biocompatibility was assessed by observing the growth of human umbilical vein endothelial cells (HUVEC) on the surface of Nitinol alloys. Static and dynamic immersion tests were performed by immersing the Nitinol alloys in cell culture media and measuring the amount of metal ions released in solution. Sulforhodamine B (SRB) assays were performed to elucidate the effect of metal ions on the growth of HUVEC cells. The surfaces of the alloys were studied using Scanning Electron Microscopy (SEM) and X-ray Photoelectron Spectroscopy (XPS) respectively. Finally, wettability and surface energy were measured by Contact Angle Meter, whereas surface roughness was measured by Atomic Force Microscopy (AFM). All the surface treated alloys exhibited high resistance to corrosion when compared with untreated alloys. SRB assays revealed that Ni and Cu ions exhibited greater toxicity than Cr, Ta and Ti ions on HUVEC cells. EP and MEP alloys possessed relatively smooth surfaces and some were composed of nickel oxides instead of elemental nickel as determined by XPS. MEP exhibited lowest surface energy and lowest surface roughness.

Biocompatibility Assessment of Biosorbable Polymer Coated Nitinol Alloys

Owing to an increased risk of aging population and a higher incidence of coronary artery disease (CAD), there is a need for more reliable and safer treatments. Numerous varieties of durable polymer-coated drug eluting stents (DES) are available in the market in order to mitigate in-stent restenosis. However, there are certain issues regarding their usage such as delayed arterial healing, thrombosis, inflammation, toxic corrosion by-products, mechanical stability and degradation. As a result, significant amount of research has to be devoted to the improvement of biodegradable polymer-coated implant materials in an effort to enhance their bioactive response. In this investigation, magneto-electropolished (MEP) and a novel biodegradable polymer coated ternary Nitinol alloys, NiTiTa and NiTiCr were prepared to study their bio and hemocompatibility properties. The initial interaction of a biomaterial with its surroundings is dependent on its surface characteristics such as, composition, corrosion resistance, work of adhesion and morphology. In-vitro corrosion tests such as potentiodynamic polarization and electrochemical impedance spectroscopic (EIS) studies were conducted to determine the coating stability and longevity. In-vitro hemocompatibility studies and HUVEC cell growth was performed to determine their thrombogenic and biocompatibility properties. Critical delamination load of the polymer coated Nitinol alloys was determined using Nano-scratch analysis. Sulforhodamine B (SRB) assays were performed to elucidate the effect of metal ions leached from Nitinol alloys on the viability of HUVEC cells. Scanning Electron Microscopy (SEM), X-ray Photoelectron Spectroscopy (XPS), contact angle meter and X-ray diffraction (XRD) were used to characterize the surface of the alloys. MEP treated and polymer coated (PC) Nitinol alloys displayed a corrosion resistant polymer coating as compared to uncoated alloys. MEP and PC has resulted in reduced Ni and Cr ion leaching from NiTi5Cr and subsequently low cytotoxicity. Thrombogenicity tests revealed significantly less platelet adhesion and confluent endothelial cell growth on polymer coated and uncoated ternary MEP Nitinol alloys. Finally, this research addresses the bio and hemocompatibility of MEP + PC ternary Nitinol alloys that could be used to manufacture blood contacting devices such as stents and vascular implants which can lead to lower U.S. healthcare spending.

A New Platinum Complex With Tryptophan: Synthesis, Structural Characterization, Dft Studies And Biological Assays In Vitro Over Human Tumorigenic Cells.

A new platinum(II) complex with the amino acid L-tryptophan (trp), named Pt-trp, was synthesized and characterized. Elemental, thermogravimetric and ESI-QTOF mass spectrometric analyses led to the composition [Pt(C11H11N2O2)2]⋅6H2O. Infrared spectroscopic data indicate the coordination of trp to Pt(II) through the oxygen of the carboxylate group and also through the nitrogen atom of the amino group. The (13)C CP/MAS NMR spectroscopic data confirm coordination through the oxygen atom of the carboxylate group, while the (15)N CP/MAS NMR data confirm coordination of the nitrogen of the NH2 group to the metal. Density functional theory (DFT) studies were applied to evaluate the cis and trans coordination modes of trp to platinum(II). The trans isomer was shown to be energetically more stable than the cis one. The Pt-trp complex was evaluated as a cytotoxic agent against SK-Mel 103 (human melanoma) and Panc-1 (human pancreatic carcinoma) cell lines. The complex was shown to be cytotoxic over the considered cells.

Liposomal Lidocaine Gel For Topical Use At The Oral Mucosa: Characterization, In Vitro Assays And In Vivo Anesthetic Efficacy In Humans.

To characterize liposomal-lidocaine formulations for topical use on oral mucosa and to compare their in vitro permeation and in vivo anesthetic efficacy with commercially available lidocaine formulations. Large unilamellar liposomes (400 nm) containing lidocaine were prepared using phosphatidylcholine, cholesterol, and α-tocoferol (4:3:0.07, w:w:w) and were characterized in terms of membrane/water partition coefficient, encapsulation efficiency, size, polydispersity, zeta potential, and in vitro release. In vitro permeation across pig palatal mucosa and in vivo topical anesthetic efficacy on the palatal mucosa in healthy volunteers (double-blinded cross-over, placebo controlled study) were performed. The following formulations were tested: liposome-encapsulated 5% lidocaine (Liposome-Lido5); liposome-encapsulated 2.5% lidocaine (Liposome-Lido2.5); 5% lidocaine ointment (Xylocaina®), and eutectic mixture of lidocaine and prilocaine 2.5% (EMLA®). The Liposome-Lido5 and EMLA showed the best in vitro permeation parameters (flux and permeability coefficient) in comparison with Xylocaina and placebo groups, as well as the best in vivo topical anesthetic efficacy. We successfully developed and characterized a liposome encapsulated 5% lidocaine gel. It could be considered an option to other topical anesthetic agents for oral mucosa.

Synthesis and biocompatibility of an experimental glass ionomer cement prepared by a non-hydrolytic sol-gel method

The aims of this study were to demonstrate the synthesis of an experimental glass ionomer cement (GIC) by the non-hydrolytic sol-gel method and to evaluate its biocompatibility in comparison to a conventional glass ionomer cement (Vidrion R). Four polyethylene tubes containing the tested cements were implanted in the dorsal region of 15 rats, as follows: GI - experimental GIC and GII - conventional GIC. The external tube walls was considered the control group (CG). The rats were sacrificed 7, 21 and 42 days after implant placement for histopathological analysis. A four-point (I-IV) scoring system was used to graduate the inflammatory reaction. Regarding the experimental GIC sintherization, thermogravimetric and x-ray diffraction analysis demonstrated vitreous material formation at 110oC by the sol-gel method. For biocompatibility test, results showed a moderate chronic inflammatory reaction for GI (III), severe for GII (IV) and mild for CG (II) at 7 days. After 21 days, GI presented a mild reaction (II); GII, moderate (III) and CG, mild (II). At 42 days, GI showed a mild/absent inflammatory reaction (II to I), similar to GII (II to I). CG presented absence of chronic inflammatory reaction (I). It was concluded that the experimental GIC presented mild/absent tissue reaction after 42 days, being biocompatible when tested in the connective tissue of rats.