Simulated Physiological Fluids Research Articles

Novel Three-Component Composite Materials (Hydroxyapatite/Polymer Mixtures) for Bone Regeneration

Biomaterial is a non-drug substance suitable for inclusion in systems which augment or replace the function of body tissues or organs. The nano-hydroxyapatite (HAp) is one of the most applied biomaterial, a popular bone substitute, for coatings and other filler materials due to their ability to promote mineralization. Hydroxyapatite [Ca10(PO4)6(OH)2] is one of the most stable forms of calcium phosphate and it occurs in bones as major component (60 to 65%), along with other materials including collagen, chondroitin sulfate, keratin sulfate and lipids. In order to improve the bioactivity and mechanical properties of hydroxyapatite, some synthetic and natural polymers (e.g., cellulose, collagen, chitosan, chitine) have been used as excellent candidates for bone/cartilage tissue engineering applications. In this paper we propose to synthesize novel three-component composite materials from HAp and cellulose (carboxymethyl cellulose, CMC) with one of the following natural polymers (chitosan (CS), chitine (CT), collagen (CLG), all of them named CX), biocompatible (from CMC) and with excellent antimicrobial activity (from HAp). Similarly to CS, CMC has an extensive network of intra- and inter-molecular hydrogen bonds which makes it insoluble in water or in common organic solvents. To prepare this new composite material, ionic liquids (ILs) have been used as potential green solvents for biopolymers, knowing that 1-butyl-3-methylimidazolium chloride [BMImCl] is able to dissolve up to 10% (w/w) of CMC, and other polysaccharides such as CS. In the studied systems, HAp particles were dispersed uniformly in organic phase, and are present strong chemical interactions between the three phases. In our case, a highly porous film was prepared and its bioactivity was investigated byin vitrotests in a simulated body fluid (SBF) for 6 months. By ICP-OES, was found a migration of analyzed metals (Fe, Mn, Cr and Ni) in simulated physiological fluids (SBF) analyzed after a period of 6 months, but only in the ppb concentrations range. Different system (HAp/CX/CMC) composites with weight ratios of 70/10/20, 70/15/15 and 70/20/10 were prepared. The new composites were characterized by infrared spectroscopy Fourier transformed (FT-IR), scanning electron microscopy (SEM) and ICP-OES. Their antimicrobial activity has been tested onCandida albicans,Candida parapsilosisandStaphylococcus aureus,proofing an excellent antimicrobial activity.

Surface analysis and electrochemical behavior of Ti–20Zr alloy in simulated physiological fluids

An advanced Ti–20Zr alloy was obtained by double vacuum melting in a semi-levitation furnace with cold crucible. The alloy shows fully lamellar α + β microstructure. Cyclic potentiodynamic polarization curves revealed that the alloy passivated easier, more rapid than Ti, having a more stable passive film in Ringer solutions of different pH values, simulating severe functional conditions of an implant. In neutral and alkaline Ringer solutions, the alloy passive film improved its properties in time (1500 h) by the deposition of protective hydroxyapatite, as was demonstrated by XPS, SEM, EDX, Raman and FT-IR measurements. Alloy presented lower corrosion rates and higher polarization resistances (from linear polarization measurements) than those of Ti (tens of times) proving a more resistant passive film. Alloy open circuit potentials had more electropositive values in comparison with Ti and tended to nobler values in time, which denote better passive state and its enhancement in time, due to the new depositions from the physiological solutions. Nyquist and Bode spectra depicted a more protective passive film on the alloy surface than on Ti surface. The passive film is formed by two layers: an inner barrier layer and an outer porous layer. An electric equivalent circuit with two time constants was modeled.

Synthesis, characterization and bioactivity of bioglasses in the Na2O–CaO–P2O5–SiO2 system prepared via sol gel processing

Different kinds of bioactive materials are used as bone substitutes. In particular, bioglasses containing Si, Ca, Na and P, bind to host tissues with a stable chemical bond. The phenomenon of bioactivity is associated with the formation of a crystallized hydroxycarbonated apatite (HCA) layer on the bioglass surface, when soaked in a simulated physiological fluid. This layer is similar to the mineral phase of bone.Synthesized bioglasses have been obtained using organic modifiers instead of mineral modifiers, which are the usual precursors for sol–gel synthesis.First, a slightly soluble ternary glass, containing 48% CaO, 5% P2O5 and 47% SiO2, referred to as Si47P5T, was prepared.Then, some sodium oxide has been added to obtain a more bioactive quaternary bioglass, containing 21.5% Na2O, 26.5% CaO, 5% P2O5 and 47% SiO2 which is referred to as Si47P5Q.Physicochemical properties of both bioglasses have been studied. Bioactivity in a simulated body fluid (SBF) and cytotoxicity tests have complemented the study.

Medicated hydrogels of hyaluronic acid derivatives for use in orthopedic field

Physical hydrogels have been obtained from hyaluronic acid derivatized with polylactic acid in the presence or in the absence of polyethylene glycol chains. They have been extemporarily loaded with antibacterial agents, such as vancomycin and tobramycin. These medicated hydrogels have been used to coat titanium disks (chosen as simple model of orthopedic prosthesis) and in vitro studies in simulated physiological fluid have been performed as a function of time and for different drug loading and polymer concentration values. Sterilization process performed on the hydrogels does not change their rheological behavior and release properties as well as the chemical structure of starting copolymers. A preliminary test has been performed by coating with the hydrogel a prosthesis that has been inserted in a seat of a lyophilized human femur, to confirm the ability of the hydrogel to adhere to the prosthesis surface also after its insertion in the implant seat. Cell compatibility of obtained hydrogels has been confirmed in vitro by using human dermal fibroblasts chosen as a model cell line. Obtained results suggest the potential use of these hydrogels in the orthopedic field, in particular for the production of antibacterial coatings of prostheses for implant in the human or animal body in the prevention and/or treatment of post surgical infections.

Electrochemical Behaviour of Ti-20Nb-10Zr-5Ta Alloy in Simulated Physiological Fluids after Anodic Electrolytic Oxidation

Electrochemical Behaviour of Ti-20Nb-10Zr-5Ta Alloy in Simulated Physiological Fluids after Anodic Electrolytic Oxidation

Electrochemical Behavior of Ti-6Al-7Nb in Simulated Physiological Body Fluid Environment

The electrochemical behavior of Ti-6Al-7Nb was investigated, and compared with that of Ti–6Al–4V alloy, which is commonly used as implant material, particularly for dentistry, orthopaedic and osteosynthesis applications. The aim of present study was to evaluate their corrosion resistance in an artificial physiological conditions using Hank’s solution at 37◦C. This evaluation was carried out through the analysis of the potentiodynamic polarization (PD), and electrochemical impedance spectroscopy (EIS) tests. Very low current densities were obtained from the PD curves, indicating a typical passive behavior for two investigated alloys. The EIS results at open circuit potential and at different potential exhibited capacitive behavior (high corrosion resistance) with phase angles close to −90◦ and high impedance values at low and medium frequencies, which are indicative of the formation of a highly stable film on these alloys in the test solution. The resistance of TAN is slightly higher than that of TAV. This difference can be attributed to the alloying elements. For TAN, the formation of Nb2O5 oxide, which is chemically more stable and less soluble compared to V2O5 formed on the Ti–6Al–4V alloy, enhances resistance to dissolution of the surface film. Thus, it has been suggested that Ti–6Al–7Nb can be a better alternative to Ti–6Al–4V in simulated body fluid environment.

Electrochemical Behavior of Ti-6Al-7Nb in Simulated Physiological Body Fluid Environment

not Available.

Self-organized nanoparticles based on drug-interpolyelectrolyte complexes as drug carriers

Potential applications in drug delivery from nanostructures composed of two oppositely charged polymethacrylates, eudragit L100 (EL) and eudragit EPO (EE), loaded with three model basic drugs (D), atenolol, propranolol, and metroclopramide were evaluated. The self-organized nanoparticles based on drug-interpolyelectrolyte complexes (DIPEC), (EL-D50)-EEX, were obtained by mixing the aqueous dispersions of both polyelectrolytes at room temper- ature in an ultrasound bath. Dispersions of (EL-D50) neutralized with increasing proportions of EE exhib- ited a rise of turbidity, particle sizes in the range of 150-400 nm, and high negative zeta potential. The sign of zeta potential was shifted from negative to positive by changes in composition of DIPEC. Freeze dried DIPEC were easily redispersed in water yielding nearly the same parameters of fresh dispersions. In vitro release experiments using Franz cells showed that DIPEC systems behave as a drug reservoir that slowly releases the drug as water is placed in the receptor compartment. The release rate was raised by ionic exchange with counterions present in simulated physiological fluids placed in the receptor media. Delivery of D from DIPEC exhibited a remarkable robustness toward simulated physiological media of different pH. The DIPEC systems exhibit interesting properties to design nanoparticulate drug delivery systems for oral and/or topical routes.

Evaluation of the interface aging process of polypyrrole–polysaccharide electrodes in a simulated physiological fluid

Composite polypyrrole–polysaccharide (PPyPSacc) films, such as polypyrrole–heparin (PPyHep), polypyrrole–chondroitin sulfate A (PPyCS-A) and polypyrrole–hyaluronic acid (PPyHA) have been successfully obtained by optimized electrochemical syntheses. The PPyPSacc samples show dopant-mediated tunable physical properties and good stability in air and in biological fluids. The immobilization of PSacc within the polymer matrix renders the surface samples suitable for cell/substrate communication studies. In order to check the viability of PPyPSacc thin films as electrodes for cell electrostimulation, we studied their interface evolution upon aging in a simulated physiological solution (pH=7.4, 37°C) by using electrochemical impedance spectroscopy, cyclic voltammetry and scanning electron microscopy. At open circuit conditions, the overall impedance of the electrodes increases upon aging as a consequence of a fast accumulation of ions at the interface, also related to the increase of porosity and wettability. As the interface impedance increases, the ionic and electronic transport into the polymer is hindered. Nevertheless, no mechanical degradation of the PPyPSacc samples is observed on aging. After 10 days of aging the PPyHep samples keep about 60% of their initial redox capacity. As a consequence the studied materials are capable to supply electrical stimulations to cells in a biological environment well beyond the conventional time-range.

NMR imaging of chitosan and carboxymethyl starch tablets: Swelling and hydration of the polyelectrolyte complex

The hydration and swelling properties of the tablets made of chitosan, carboxymethyl starch, and a polyelectrolyte complex of these two polysaccharides have been studied by NMR imaging. We studied the effect of pH and ionic strength on the swelling of the tablets and on the diffusion of fluid into the tablets in water and simulated physiological fluids. The pH value of the fluids exerts a more significant effect than their ionic strengths on the swelling of the tablets. The tablets are compared also with those made of cross-linked high amylose starch. The formation of complex helps to keep the integrity of the tablets in various media and render a slow and restricted swelling similar to that of the tablets of the cross-linked high amylase starch, which is significantly lower than the swelling of chitosan and of carboxymethyl starch. The capacities to modulate the release rate of drugs in different media are discussed by comparing the matrices and evaluating the preparation process of the complex. A sustained release of less soluble drugs such as aspirin in gastrointestinal fluids can be provided by the complex, due to the ionic interaction and hydrogen bonding between the drug and the biopolymer complex.

Interaction between Drug Loaded Polyaspartamide-Polylactide-Polysorbate Based Micelles and Cell Membrane Models: A Calorimetric Study

Amphiphilic biodegradable copolymers, for their ability to self-assemble into micelle-like aggregates, with a suitable loading capacity, are of emerging interest for the delivery of water-insoluble drugs. α,β-Poly[(N-hydroxyethyl)-dl-aspartamide] (PHEA) is suitable to obtain amphiphilic graft copolymers. These copolymers can be obtained starting from PHEA-ethylenediamine (PHEA-EDA) which is functionalized with polysorbate 80 (PS₈₀, like targeting residues to the brain) and polylactide (PLA, like hydrophobic chains) in order to obtain polymeric micelles of PHEA-EDA-PS₈₀-PLA potentially useful to release drugs to the central nervous system. In this paper, the interaction and absorption of PHEA-EDA-PS₈₀-PLA micelles loaded with (R)-flurbiprofen with biomembrane models, represented by multilamellar or unilamellar vesicles made of dimyristoylphosphatidylcholine, are investigated by means of differential scanning calorimetry technique. (R)-Flurbiprofen is the single enantiomer of the racemate flurbiprofen; the capacity of this nonsteroidal anti-inflammatory drug to reduce risk of Alzheimer's disease has been recently reported. Drug release from the micelles to the lipid vesicles has been investigated in simulated physiological fluid, and it resulted to be affected by the biomembrane model.

Biocompatible hydrogels based on hyaluronic acid cross-linked with a polyaspartamide derivative as delivery systems for epithelial limbal cells

The aim of this work was to evaluate the potential use of hydrogels based on hyaluronic acid (HA) chemically cross-linked with α,β-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)- d, l-aspartamide (PHEA-EDA) as substitutes for the amniotic membrane able to release limbal cells for corneal regeneration. Hydrogels, shaped as films, with three different molar ratios ( X) between PHEA-EDA and HA ( X = 0.5, 1.0 and 1.5) have been investigated. First, it has been evaluated their swelling ability, hydrolytic resistance in simulated physiological fluid and cell compatibility by using human dermal fibroblasts chosen as a model cell line. Then adhesion studies in comparison with collagen gel, have been performed by using immortalized cells, such as human corneal epithelial cells (HCEC) or primary cells, such as rabbit limbal epithelial cells (RLEC) and/or rabbit limbal fibroblasts (RLF). HA/PHEA-EDA hydrogels allow a moderate/poor adhesion of all investigated cells thus suggesting their potential ability to act as cell delivery systems. Finally, commercial contact lenses have been coated, in their inner surface, with each HA/PHEA-EDA film and it has been found that in these conditions, a greater cell adhesion occurs, particularly when RLEC are in co-culture with RLF. However, this adhesion is only transitory, in fact after three days, viable cells are released in the culture medium thus suggesting a potential application of HA/PHEA-EDA hydrogels, for delivering limbal cells in the treatment of corneal damage.

Bioavailability of lycopene in the rat: the role of intestinal lymphatic transport

As a natural antioxidant derived from dietary sources, lycopene has attracted considerable attention as a potent chemopreventative agent. Lycopene is an extremely lipophilic compound and absorption from dietary sources is estimated to be low and highly variable. As a result, plasma lycopene concentrations are poorly correlated with dietary intake of lycopene rich food stuffs. The development of an oral formulation remains a challenge that requires a better understanding of the mechanisms involved in the intestinal absorption of this compound. The solubility of lycopene in simulated physiological fluids and bile salt mixed micelle formulations was determined. The extent of intestinal lymphatic transport and the absolute bioavailability of lycopene from a range of biorelevant media was evaluated in a mesenteric lymph duct cannulated anaesthetised rat model. The absolute bioavailability of lycopene after 8 h was 1.85 +/- 0.39%. The overall extent of the intestinal lymphatic transport was in the range of 0.6-3.4% of the administered dose. A strong positive correlation (r(2) > 0.9) between intestinal lycopene levels and intestinal triglyceride levels was demonstrated. The intestinal lymphatic route is the major uptake mechanism of lycopene from the gastrointestinal tract. Lycopene transport in intestinal lymph was closely associated with triglyceride transport in the lymph. Formulation strategies designed to promote intestinal lymphatic uptake, such as lipid-based formulations containing long-chain fatty acids (LCFA) or lecithin, may serve to enhance oral bioavailability of lycopene.

Mystery of the transformation from amorphous calcium phosphate to hydroxyapatite

The process from the amorphous calcium phosphate phase to the hydroxyapatite (HAP) crystalline phase has been captured in a simulated physiological fluid by combining in situ extinction detection and ex situ electronic microscopy. The results reveal the secret of the phase transformation and orientation controls during the initial stage of mineral formation.

Nanodiamond-based tribosystems

The tribological behaviour of homologous contacts between nanocrystalline diamond (NCD) chemically vapour deposited (CVD) coated parts is reviewed. The major part of the published works is related to silicon nitride ceramic coated materials. The NCD-on-NCD tribo-tests were performed under unlubricated and lubricated sliding conditions in water and in simulated physiological fluids (Hank's balanced salt solution, HBSS, and dilute fetal bovine serum, FBS). The friction and wear behaviour was assessed using a reciprocating motion setup under moderated to high applied normal loads (10–90 N), and up to 6 km of sliding distance. The inherent very low surface roughness of the NCD films was responsible for a very low steady-state friction response (0.01–0.05) in unlubricated and water-lubricated tests. Moreover, the running-in period of accommodation observed for conventional microcrystalline diamond coatings is greatly suppressed by the much lower starting surface roughness found in the NCD coatings. In distilled water, the NCD coatings displayed a remarkable tribological performance characterised by a high resistance to wear damage (10 − 9 mm 3 N − 1 m − 1 ), characteristic of very mild wear regimes, and high tribomechanical threshold loads (85 N) before film failure. Biotribological experiments conducted in HBSS resulted in similar friction and wear coefficients, while tribotesting in FBS revealed higher friction values (0.10) due to a protein attaching effect.

Novel core-shell lipid-chitosan and lipid-poloxamer nanocapsules: stability by hydration forces

In the last few years, a great attention has been paid to core-shell structured lipid nanocapsules. The interest of these colloids lies in their very promising applications as delivery systems of hydrophobic drugs or as DNA carriers. The aim of this work was to provide fuller knowledge concerning the physicochemical properties of these new colloidal particles, paying special attention to the role played by the constituting components. Our lipid nanocapsules were comprised of a triglyceride-lecithin core surrounded by a chitosan and/or poloxamer (Pluronic® F68) shell. Four different systems were formulated by varying the chitosan and poloxamer contents. We have estimated their shell composition from electrokinetic mobility measurements and stability studies. They revealed a high incorporation of chitosan to the shell, while Pluronic® F68 only presented a secondary role during the nanocapsule formation, providing final systems with low poloxamer content. In addition, the stability studies, performed not only on ideal solutions but also on simulated physiological fluids, revealed that hydration forces were crucial to maintain the integrity of these nanocapsules under physicochemical conditions similar to those found in real physiological fluids. Theoretical treatment of stability data has allowed us to determine the role that ions play on hydration forces when they act as counterions.

Biomimetic nanostructured coatings on nano-grained/ultrafine-grained substrate: Microstructure, surface adhesion strength, and biosolubility

We describe here the structural morphology, nucleation and growth, surface adhesion response and biosolubility in simulated physiological fluid of nanohydroxyapatite (nHA)-based coatings (nHA, chitosan-nHA, collagen-nHA) on nano-grained (NG)/ultrafine-grained (UFG) 316L austenitic stainless steel bulk substrate. The NG/UFG stainless steel was processed by a novel procedure involving controlled phase reversion of strain-induced martensite. Electron microscopy studies indicated that the morphology of nHA on NG/UFG stainless steel is characterized by a vein-type interconnected structure consisting of fibrils that closely mimics the fibrous structure present as part of the hierarchical structure of the bone. Interestingly, the dimensions of the vein-type interconnected structure were similar to the grain size of NG/UFG stainless steel substrate. This structural morphology was retained in crystallized chitosan-nHA coating, but was modified when collagen is codeposited with nHA. The mechanisms of deposition are discussed based on the structural characteristics of coatings. Adhesion response of coating as determined by scratching tests suggested superior adhesion strength of nHA coating on NG/UFG substrate as compared to that on CG substrate and is attributed to grain size effect and wettability of the substrate.

A resorbable calcium-deficient hydroxyapatite hydrogel composite for osseous regeneration

It was previously discovered that the unique structure and chemistry of bacterial cellulose (BC) permits the formation of calcium-deficient hydroxyapatite (CdHAP) nanocrystallites under aqueous conditions at ambient pH and temperature. In this study, BC was chemically modified via a limited periodate oxidation reaction to render the composite degradable and thus more suitable for bone regeneration. While native BC does not degrade in mammalian systems, periodate oxidation yields dialdehyde cellulose which breaks down at physiological pH. The composite was characterized by tensile testing, X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy. X-ray diffraction showed that oxidized BC retains its structure and could biomimetically form CdHAP. Degradation behavior was analyzed by incubating the samples in simulated physiological fluid (pH 7.4) at 37 °C under static and dynamic conditions. The oxidized BC and oxidized BC-CdHAP composites both lost significant mass after exposure to the simulated physiological environment. Examination of the incubation solutions by UV–Vis spectrophotometric analysis demonstrated that, while native BC released only small amounts of soluble cellulose fragments, oxidized cellulose releases carbonyl containing degradation products as well as soluble cellulose fragments. By entrapping CdHAP in a degradable hydrogel carrier, this composite should elicit bone regeneration then resorb over time to be replaced by new osseous tissue.

Bioinspired mineralization and cell adhesion on surface functionalized poly(vinyl alcohol) films

Poly(vinyl alcohol) (PVA) films, when surface functionalized by phosphorylation, induced biomimetic nucleation and growth of calcium phosphate in a simulated physiological environment. The surface phosphorylation on PVA was ensured by attenuated total reflectance infrared spectroscopy. The morphology of the calcium phosphate phase grown on surface-phosphorylated PVA (PPVA) was analysed using scanning electron microscopy coupled with an energy-dispersive X-ray detector. The primary nucleation of calcium phosphate occurs in 3 days and secondary nucleation occurs after 10 days. The energy-dispersive X-ray analysis shows that the Ca/P ratio of the coating increases with time of exposure to the simulated physiological fluid and reaches 1.67 at 10 days. The PPVA supports in vitro cell adhesion and promotes in vitro biomineralization in the presence of cells, evaluated using human osteosarcoma cells.

The molecular structure of complexes formed by chromium or cobalt ions in simulated physiological fluids

It is well recognized that workers in mines and manufacturers of hard metals in contact with metal ions have increased risk of cancer. However, the various chemical forms of these ions in living organisms are not well characterized, and little is known of how they interact with biological environments. Here, we sought to elucidate the molecular structures formed by cobalt (Co) and chromium (Cr) ions in simulated biological fluids. Transmission electron microscopy observations revealed that Cr(III) formed nanoscale complexes which precipitate in both RPMI 1640 and DMEM high glucose media. However, no complexes were observed with Co(II) and Cr(VI). Energy-dispersive X-ray analysis, elemental analysis, and Fourier transform infrared (FT-IR) spectroscopy indicated that Cr(III) ions act as chelating entities between PO 4 groups, amino acids, and calcium. Although the exact nature of the bonds remains to be investigated, the presence of PO 4 may favor the formation of low energy hydrogen bonds. Interestingly, the nature of amino acids varied in Cr(III) complexes formed in RPMI 1640 compared to those formed in DMEM high glucose. The absence of sulphur in the elemental analysis spectrum suggested that methionine and cystine, two amino acids containing sulphur, are not involved in the formation of Cr(III) complexes. Thus, the lower toxicity of Cr(III) compared to Co(II) and Cr(VI) may be related to the formation of these chemical complexes. These results may bring some insight in understanding the relationship between toxicity and the chelating capabilities of various metal ions in vitro and in vivo.