Physiological-like Solution Research Articles

1,3,5-Triaza-7-phosphaadamantane and Cyclohexyl Groups Impart to Di-Iron(I) Complex Aqueous Solubility and Stability, and Prominent Anticancer Activity in Cellular and Animal Models.

Using a multigram-scalable synthesis, we obtained nine dinuclear complexes based on nonendogenous iron(I) centers and featuring variable aminocarbyne and P-ligands. One compound from the series (FEACYP) emerged for its strong cytotoxicity in vitro against four human cancer cell lines, surpassing the activity of cisplatin by 3-6 times in three cell lines, with an average selectivity index of 6.2 compared to noncancerous HEK293 cells. FEACYP demonstrated outstanding water solubility (15 g/L) and stability in physiological-like solutions. It confirmed its superior antiproliferative activity when tested in 3D spheroids of human pancreatic cancer cells and showed a capacity to inhibit thioredoxin reductase (TrxR) similar to auranofin. In vivo treatment of murine LLC carcinoma with FEACYP (8 mg kg-1 dose) led to excellent tumor growth suppression (88%) on day 15, with no signs of systemic toxicity and only limited body weight loss.

Bond strength between dentine and a novel fast-setting calcium silicate cement with fluoride.

The aim of this study was to evaluate the dentine bond strength of a novel fast-setting calcium silicate cement (Protooth) versus a calcium hydroxide-based cement (Dycal), a calcium silicate cement (ProRoot MTA), and a glass ionomer cement (Ketac-Molar). Mid-root dentine slices of 1mm thickness were obtained from human maxillary incisors. After enlarging the lumen of the canal to 1.3mm, the cavities were randomly filled with test materials. Samples were immersed in physiological-like solution. The push-out bond strength was tested on days 1, 28, and 180 (n=12). Failure types of bonding were determined using a stereomicroscope. We analysed the data using linear regression. Dycal and day 1 were considered as reference for cement type and assessment time, respectively. Protooth, Ketac-Molar, and ProRoot MTA demonstrated higher push-out bond strength than Dycal. The push-out bond strength in the Protooth group increased on day 28 and 180. The bond strength of Ketac-Molar was significantly reduced on day 28. Dycal showed a significant decrease in bond strength on day 180 compared with that on day 1 and 28. Mixed failure was the dominant failure type. Protooth bonding to dentine was increased with time, in contrast to that of ProRoot MTA, Dycal, and Ketac-Molar, as a function of time.

Particulate metal bioaccessibility in physiological fluids and cell culture media: Toxicological perspectives

According to the literature, tiny amounts of transition metals in airborne fine particles (PM2.5) may induce proinflammatory cell response through reactive oxygen species production. The solubility of particle-bound metals in physiological fluids, i.e. the metal bioaccessibility is driven by factors such as the solution chemical composition, the contact time with the particles, and the solid-to-liquid phase ratio (S/L). In this work, PM2.5-bound metal bioaccessibility was assessed in various physiological-like solutions including cell culture media in order to evidence the potential impact on normal human bronchial epithelial cells (NHBE) when studying the cytotoxicity and inflammatory responses of PM2.5 towards the target bronchial compartment. Different fluids (H2O, PBS, LHC-9 culture medium, Gamble and human respiratory mucus collected from COPD patients), various S/L conditions (from 1/6000 to 1/100,000) and exposure times (6, 24 and 72h) were tested on urban PM2.5 samples. In addition, metals’ total, soluble and insoluble fractions from PM2.5 in LHC-9 were deposited on NHBE cells (BEAS-2B) to measure their cytotoxicity and inflammatory potential (i.e., G6PDH activity, secretion of IL-6 and IL-8). The bioaccessibility is solution-dependent. A higher salinity or organic content may increase or inhibit the bioaccessibiliy according to the element, as observed in the complex mucus matrix. Decreasing the S/L ratio also affect the bioaccessibility depending on the solution tested while the exposure time appears less critical. The LHC-9 culture medium appears to be a good physiological proxy as it induces metal bioaccessibilities close to the mucus values and is little affected by S/L ratios or exposure time. Only the insoluble fraction can be linked to the PM2.5-induced cytotoxicity. By contrast, both soluble and insoluble fractions can be related to the secretion of cytokines. The metal bioaccessibility in LHC-9 of the total, soluble, and insoluble fractions of the PM2.5 under study did not explain alone, the cytotoxicity nor the inflammatory response observed in BEAS-2B cells. These findings confirm the urgent need to perform further toxicological studies to better evaluate the synergistic effect of both bioaccessible particle-bound metals and organic species.

Rheological and Morphological Investigation of Renaturated Collagen Nanogels in Physiological-like Solution Conditions

Collagen is the most abundant protein in mammals comprising 70% of skin, tendons and bones. Denaturated (or renaturated) collagen is also referred to as gelatin. Gelatin gels are made up of a 3D network of renaturated collagen nanofibres and are one of the most frequently used protein-based materials in a wide range of industrial applications; for this reason they attract a strong interest from the international scientific and industrial communities. Applications of gelatin gels in biomedical engineering, although widely spread, are still hampered by the apparent instability of the gelatin network under physiological conditions. This matter gets further complicated by the confusion surrounding the usage of different types of gelatin as well as the, usually overseen, potential effects of a variety of different experimental parameters on the final gel properties. Here we present the first of a series of detailed studies in which we begin to investigate the rheological and structural properties of gelatin type B in an increasingly more biologically relevant and complex solution-environment. Protein concentration, pH, ionic strength and environmental temperature can strongly affect the gel formation capacity of collagen. Here we begin to investigate the effects of the above mentioned parameters on nanogels of gelatin type B. Biomechanical temperature ramp step tests were carried out in order to determine the melting (or formation) temperatures of the gels whilst electron microscopy probed the structural properties of the underlying nanostructures. We believe that systematic investigations can lead us to gain a deeper insight into these popular biomaterials with the view of widening and further facilitating their applications in biomedical engineering.

Improving mobility and electrochemical stability of a water-gated polymer field-effect transistor

Water-gated organic transistors have attracted considerable attention in the field of biosensors, thanks to their capability of operating in the aqueous environment typical of biological systems at very low voltages (∼1V). Some examples have been recently reported in the literature, employing different organic materials as the active semiconducting layer, ranging from small molecules to single crystals. Here we report on water-gated polymer-based organic-field effect devices using poly(2,5-bis(3-hexadecylthiophen-2-yl)thieno[3,2-b]thiophene) (pBTTT) as the active layer. Very promising electronic performances, in terms of mobility and operating voltages are obtained; notably, the charge carrier mobility is in the order of 0.08cm2/Vs, which is of the same order of magnitude of values reported for single-crystal based water-gated devices, and consistent with values reported for solid-state polymer dielectric transistors. Moreover, the pBTTT-based device shows improved electrochemical stability, as compared to previously reported polymer based water-gated devices. Importantly, good functioning of the device is demonstrated also when water is replaced by physiological-like solutions. Critical to the transistors operation, besides the good transport properties of the active material, is the key-role played by alkyl side chains and ordered morphology of the polymer at the interface with the liquid environment, which we highlight here for the first time. Our contribution overall provides a useful step towards the development of bio-organic sensors, with enhanced properties in terms of sensitivity and stability, and for a successful exploitation of organic based field effect transistors in biotic/abiotic interfaces.

Biomimetic magnesium–carbonate-apatite nanocrystals endowed with strontium ions as anti-osteoporotic trigger

The present work investigates the preparation of biomimetic nanocrystalline apatites co-substituted with Mg, CO3 and Sr to be used as starting materials for the development of nanostructured bio-devices for regeneration of osteoporotic bone. Biological-like amounts of Mg and CO3 ions were inserted in the apatite structure to mimic the composition of bone apatite, whereas the addition of increasing quantities of Sr ions, from 0 up to 12wt.%, as anti-osteoporotic agent, was evaluated. The chemical–physical features, the morphology, the degradation rates, the ion release kinetics as well as the in vitro bioactivity of the as-prepared apatites were fully evaluated. The results indicated that the incorporation of 12wt.% of Sr can be viewed as a threshold for the structural stability of Mg–CO3-apatite. Indeed, incorporation of lower quantity of Sr did not induce considerable variations in the chemical structure of Mg–CO3-apatite, while when the Sr doping extent reached 12wt.%, a dramatically destabilizing effect was detected on the crystal structure thus yielding alteration of the symmetry and distortion of the PO4. As a consequence, this apatite exhibited the fastest degradation kinetic and the highest amount of Sr ions released when tested in physiological conditions. In this respect, the surface crystallization of new calcium phosphate phase when immersed in physiological-like solution occurred by different mechanisms and extents due to the different structural chemistry of the variously doped apatites. Nevertheless, all the apatites synthesized in this work exhibited in vitro bioactivity demonstrating their potential use to develop biomedical devices with anti-osteoporotic functionality.

Matrix Disruptions, Growth, and Degradation of Cartilage with Impaired Sulfation

Diastrophic dysplasia (DTD) is an incurable recessive chondrodysplasia caused by mutations in the SLC26A2 transporter responsible for sulfate uptake by chondrocytes. The mutations cause undersulfation of glycosaminoglycans in cartilage. Studies of dtd mice with a knock-in Slc26a2 mutation showed an unusual progression of the disorder: net undersulfation is mild and normalizing with age, but the articular cartilage degrades with age and bones develop abnormally. To understand underlying mechanisms, we studied newborn dtd mice. We developed, verified and used high-definition infrared hyperspectral imaging of cartilage sections at physiological conditions, to quantify collagen and its orientation, noncollagenous proteins, and chondroitin chains, and their sulfation with 6-μm spatial resolution and without labeling. We found that chondroitin sulfation across the proximal femur cartilage varied dramatically in dtd, but not in the wild type. Corresponding undersulfation of dtd was mild in most regions, but strong in narrow articular and growth plate regions crucial for bone development. This undersulfation correlated with the chondroitin synthesis rate measured via radioactive sulfate incorporation, explaining the sulfation normalization with age. Collagen orientation was reduced, and the reduction correlated with chondroitin undersulfation. Such disorientation involved the layer of collagen covering the articular surface and protecting cartilage from degradation. Malformation of this layer may contribute to the degradation progression with age and to collagen and proteoglycan depletion from the articular region, which we observed in mice already at birth. The results provide clues to in vivo sulfation, DTD treatment, and cartilage growth.

An Octacalcium Phosphate Forming Cement.

The osteoconductive and possibly osteoinductive characteristics of OCP increased the interest in preparation of bone graft materials that contain OCP in its composition. Calcium phosphate cements (CPCs) were prepared using a mixture of α-tricalcium phosphate (α-TCP) and dicalcium phosphate anhydrous (DCPA), with α-TCP/DCPA molar ratio of 1/1 and distilled water or 0.5 mol/L phosphate aqueous solution (pH = 6.1 ± 0.1) as the cement liquid. Hardening time was (30 ± 1) min for the CPC mixed with water and (5 ± 1) min for the CPC mixed with phosphate solution. Diametral tensile strength (DTS), porosity (P), and phase composition (powder x-ray diffraction) were determined after the hardened specimens had been immersed in a physiological-like solution (PLS) for 1 d, 3 d, and 7 d. In CPC specimens prepared with water, calcium hydroxyapatite (HA) was formed and DTS and P were (9.03 ± 0.48) MPa and (37.05 ± 0.20) vol % after 1 d, respectively, and (9.15 ± 0.45) MPa and (37.24 ± 0.63) vol % after 3 d, respectively. In CPC specimens prepared with phosphate solution OCP and HA were formed and DTS and P were (4.38 ± 0.49) MPa and (41.44 ± 1.25) vol % after 1 d, respectively,(4.38 ± 0.29) MPa and (42.52 ± 2.15) vol % after 3 d, respectively, and (4.30 ± 0.60) MPa and (41.38 ± 1.65) vol % after 7 d, respectively. For each group DTS and P did not change with PLS immersion time. DTS was significantly higher and P was significantly lower for CPCs prepared with water. HA formation slightly increased with immersion time from 40 mass % after 1 d to 50 mass % after 3 d in CPCs prepared with water. OCP + HA formation increased with immersion time from 30 mass % after 1 d to 35 mass % after 3 d and to 45 mass % after 7 d in CPCs prepared with 0.5 mol/L phosphate solution.

Properties of Calcium Phosphate Cements With Different Tetracalcium Phosphate and Dicalcium Phosphate Anhydrous Molar Ratios

Calcium phosphate cements (CPCs) were prepared using mixtures of tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA), with TTCP/DCPA molar ratios of 1/1, 1/2, or 1/3, with the powder and water as the liquid. Diametral tensile strength (DTS), porosity, and phase composition (powder x-ray diffraction) were determined after the set specimens have been immersed in a physiological-like solution (PLS) for 1 d, 5 d, and 10 d. Cement dissolution rates in an acidified PLS were measured using a dual constant composition method. Setting times ((30 ± 1) min) were the same for all cements. DTS decreased with decreasing TTCP/DCPA ratio and, in some cases, also decreased with PLS immersion time. Porosity and hydroxyapatite (HA) formation increased with PLS immersion time. Cements with TTCP/DCPA ratios of 1/2 and 1/3, which formed calcium-deficient HA, dissolved more rapidly than the cement with a ratio of 1/1. In conclusion, cements may be prepared with a range of TTCP/DCPA ratios, and those with lower ratio had lower strengths but dissolved more rapidly in acidified PLS.

Cartilage Aggrecan Can Undergo Self-Adhesion

Here it is reported that aggrecan, the highly negatively charged macromolecule in the cartilage extracellular matrix, undergoes Ca 2+-mediated self-adhesion after static compression even in the presence of strong electrostatic repulsion in physiological-like solution conditions. Aggrecan was chemically end-attached onto gold-coated planar silicon substrates and gold-coated microspherical atomic force microscope probe tips (end radius R ≈ 2.5 μm) at a density (∼40 mg/mL) that simulates physiological conditions in the tissue (∼20–80 mg/mL). Colloidal force spectroscopy was employed to measure the adhesion between opposing aggrecan monolayers in NaCl (0.001–1.0 M) and NaCl + CaCl 2 ([Cl −] = 0.15 M, [Ca 2+] = 0 – 75 mM) aqueous electrolyte solutions. Aggrecan self-adhesion was found to increase with increasing surface equilibration time upon compression (0–30 s). Hydrogen bonding and physical entanglements between the chondroitin sulfate-glycosaminoglycan side chains are proposed as important factors contributing to aggrecan self-adhesion. Self-adhesion was found to significantly increase with decreasing bath ionic strength (and hence, electrostatic double-layer repulsion), as well as increasing Ca 2+ concentration due to the additional ion-bridging effects. It is hypothesized that aggrecan self-adhesion, and the macromolecular energy dissipation that results from this self-adhesion, could be important factors contributing to the self-assembled architecture and integrity of the cartilage extracellular matrix in vivo.

Anionic effects on the size and shape of apatite crystals grown from physiological solutions.

Comparatively little is known of the role tissue fluid electrolytes have in establishing the size and shape of apatite crystals deposited in skeletal tissues. In vitro accretion experiments using synthetic apatite seed crystals comparable in size to bone apatite were performed to assess the extent to which these crystalline features may be affected by direct electrolyte/mineral interactions. A constant composition method was used to maintain the accretion reactions under physiological-like solution conditions (1.33 mmol/liter Ca(2+), 1.0 mmol/liter total inorganic phosphate, (0 or 26) mmol/liter carbonate, 270 mmol/kg osmolality, pH 7.4, 37 degrees C). When the mass of the new accretions equaled the initial seed mass, the solids were harvested and the net change in crystal size resulting from the new accretions was assessed by X-ray diffraction line-broadening analysis. All the electrolytes examined in this study inhibited the accretion rate. The order of effectiveness was phosvitin > polyaspartate approximately polyglutamate > 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) > bovine serum albumin (BSA) > citrate. Citrate and BSA also reduced the mean crystal size of the harvested solids compared with those harvested in the absence of added electrolyte, a finding that suggests that citrate and BSA suppressed growth of the seed crystals in favor of the proliferation of new smaller crystals. In contrast, a net increase in size following accretion compared with controls suggests that the other more strongly inhibiting electrolytes stimulated growth of the primary seed crystals and/or of the secondary crystals. These size changes, however, were anisotropic, with the anions effecting primarily increases in crystal width/thickness rather than in length. The effects were also more pronounced in the presence of carbonate. Our findings suggest that the strength of the interaction with the crystal surface may be relatively more important than molecular size or conformational complexity in establishing the effect that electrolytes have on apatite growth and proliferation. The results also suggest that adsorbed electrolytes may be a significant factor in controlling the size of apatite crystals in skeletal tissues by inducing proliferation of new crystals and/or affecting crystal shape by selectively modifying growth of the lateral dimensions.

Behavior of a calcium phosphate cement in simulated blood plasma in vitro

Objectives. The purpose of this study was to gain a better understanding of the integration of calcium phosphate cement (CPC) implants in biological tissue. Methods. An in vitro continuous flow system was employed to examine the protracted behavior of disc-shaped specimens of this bioactive material under sustained physiological-like solution conditions. Weight measurement, diametral tensile strength measurement (DTS), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and powder x-ray diffraction (XRD) were used to characterize the CPC samples as a function of immersion time. Results. When CPC was immersed in simulated blood plasma in which the Ca (2.5 mmol/L) and inorganic phosphate (1.0 mmol/L levels were kept constant, both the weight and DTS of the specimens steadily increased to about 1.5 times their original values over a period of 20 wk. SEM obsevations showed new precipitate formations in intimate contact with the original CPC surface. FTIR and XRD analyses revealed that the precipitate was a B-type carbonate hydroxyapatite (OHAp), the type of OHAp observed in bone and dentin. On the other hand, the interior of CPC discs did not show an increase in either bulk density or OHAp content. Thus, the increases in weight and DTS are attributal to the OHAp precipitation on the CPC surface. Significance. The results suggest that under in vivo conditions, CPC implants would not dissolve in physiological fluids. OHAp coatings may form on the implants, which may enhance bonding of implants to bone by mechanically strengthening the interface between them.

On the threshold frequency of long-range interactions in physiological solutions

An electrolyte solution changes with frequency from conducting to dielectric behaviour. A threshold frequency is found above which the long-range electrical interactions become effective. We report a direct experimental determination of this Maxwell frequency for aqueous salt solutions in a range of concentrations, including physiological-like solutions.