Biomimetic organic-inorganic nanocomposite coatings for titanium implants. In vitro and in vivo biological testing.

Recently described organic-inorganic nanocomposite coatings of the chemical composition: (PLL/PGA)(10)CaP[(PLL/PGA)(5)CaP](4) (coating A) and (PLL/PGA)(10)CaP[(PLL/PGA)(5)CaP](4)(PLL/PGA)(5) (coating B), applied to chemically etched titanium plates, have been tested by extensive cell culture tests and in vivo biological experiments, with uncoated titanium plates serving as controls. Before testing, coated samples were stored for extended periods of time (from 2 weeks to 8 months) under dry, sterile conditions. Cells of the cell-lines MC3T3-E1 and/or SAOS-2 were used for the following cell culture tests: initial adhesion (4 h) and proliferation (up to 21 days), cell activity (XTT test), morphology, synthesis of collagen type I and alkaline phosphatase activity (all incubation up to 21 days). In addition, coating B was tested against uncoated control in a validated in vivo pull-out model in rabbit tibia. The results of both in vitro and in vivo experiments show excellent biological properties of chemically etched titanium which are even surpassed by surfaces covered with coating B. Thus, after 8 weeks of healing the implants coated with B were significantly better attached to the cortical bone of rabbit thibiae than uncoated titanium controls with more than twice the force needed to detach coated implants. However, coating A (top crystal layer) had an adverse effect on both cell proliferation and activity, which is explained by morphological observations, showing inhibited spreading of the cells on its rough surfaces. The results also show the remarkable stability of the coatings when shelved under dry and sterile conditions.

The influence of surface active molecules on the crystallization of biominerals in solution.

In the following article studies pertaining to "in situ" interactions of growing biogenic crystals (calcium phosphates, carbonates and oxalates) with, soluble, surface active molecules, including small, highly charged organic molecules, natural and synthetic polymers and synthetic surfactants, are discussed. Such interactions are at the roots of crystallization processes occurring in nature (biological mineralization) and in the controlled production of materials with well defined crystal structure, morphology and phase composition. The main characteristics of the crystals, including crystallographic data, and of the organic molecules, including their molecular structures, are briefly described. Most of the model crystals are crystal hydrates, whose dominant crystal planes are covered with continuous layers of structural water molecules (hydrated layer). The experimental methods reviewed include kinetic experiments determining induction times and/or the rates and rate controlling mechanisms of seeded and unseeded crystallization, techniques for the characterization of the nascent solid phase(s), and techniques, suitable for the assessment of interactions on the molecular level. Numerous examples show that the dominant mechanism underlying host crystal/additive interactions is selective adsorption of the additive at the crystal/solution interface, with the main driving forces ranging from purely electrostatic to highly specific recognition of crystal faces by the additive. Selective electrostatic interactions take place between growing crystals and flexible, highly charged small and macromolecules and/or surfactants because of differing ionic structures and charges of the crystal planes, some of them being shielded by hydrated layers. As in solution, surfactant molecules at high concentrations self-assemble into various superstructures (hemimicelles, bilayers) at the crystal/solution interface. Recognition of crystal planes by rigid small molecules and macromolecules with partial beta-sheet conformation (such as proteins or polyelectrolytes) is highly specific. It requires a dimensional fit between the distances of constituent ions protruding from the affected crystal plane(s) and the distances between functional groups that are part of the additive molecules. The consequences of selective additive/crystal interactions range from changes in crystal growth morphology to changes in the composition of the crystallizing phase. Examples showing the dual role of macromolecules as initiators and retarders of crystallization are discussed.

Interactions in Calcium Oxalate Hydrate/Surfactant Systems.

Phase transformation of calcium oxalate dihydrate (COD) into the thermodynamically stable monohydrate (COM) in anionic (sodium dodecyl sulfate (SDS)) and cationic (dodecylammonium chloride) surfactant solutions has been studied. Both surfactants inhibit, but do not stop transformation from COD to COM due to their preferential adsorption at different crystal faces. SDS acts as a stronger transformation inhibitor. The general shape of adsorption isotherms of both surfactants at the solid/liquid interface is of two-plateau-type, but differences in the adsorption behavior exist. They originate from different ionic and molecular structures of crystal surfaces and interactions between surfactant headgroups and solid surface. Copyright 1999 Academic Press.

Effect of Tamm-Horsfall protein on calcium oxalate precipitation.

The effect of Tamm-Horsfall protein isolated from urine of healthy subjects on calcium oxalate precipitation was studied in model systems of precipitation. The study was performed using following conditions: concentrations of calcium chloride 10 mmol/l, sodium chloride 150 mmol/l, oxalic acid 300 mumol/l; pH 6.0, and temperature 310 K. The concentration of Tamm-Horsfall protein varied between 1-10 mg/l. The kinetics of calcium oxalate precipitation was observed by measuring the number and volume of particles in the suspension, and the precipitate composition by an optic microscope. In all the studied systems, the precipitate morphology corresponded to pure calcium oxalate monohydrate. Tamm-Horsfall protein was found to inhibit the growth of calcium oxalate monohydrate crystals and stimulate their aggregation in the given experimental conditions. Both effects were enhanced by increase in the concentrations of Tamm-Horsfall protein and were most pronounced at the concentration of Tamm-Horsfall protein of 10 mg/l.

Induction of crystallization of calcium oxalate dihydrate in micellar solutions of anionic surfactants.

Calcium oxalate dihydrate (CaC2O4 center dot (2+x)H2O; COD; x < or = 0.5) does not readily crystallize from electrolytic solutions but appears as a component in crystalluria. In this paper, we review in vitro studies on the factors responsible for its nucleation and growth with special attention given to the role of surfactants. The following surfactants were tested: dodecyl ammonium chloride (cationic), octaethylene mono-hexadecylether (non-ionic), sodium dodecyl sulfate (SDS, anionic), dioctyl sulphosuccinate (AOT, anionic), and sodium cholate (NaC, anionic). The cationic and some of the anionic surfactants (SDS, AOT) induced different habit modifications of growing calcium oxalate crystals by preferential adsorption at different crystal faces. In addition, the anionic surfactants effectively induced crystallization of COD at the expense of COM, the proportion of COD in the precipitates abruptly increasing above a critical surfactant concentration, close to, but not necessarily identical with the respective CMC. A mechanism is proposed, whereby crystallization of COD in the presence of surfactants is a consequence of the inhibition of COM by preferential adsorption of surfactant hemimicelles (two-dimensional surface aggregates) at the surfaces of growing crystals.

Interactions of matrix proteins from mineralized tissues with octacalcium phosphate.

Acidic matrix macromolecules, present in many mineralized tissues, including those of vertebrates, are thought to be involved in controlling crystal formation. Little, however, is known about their in vivo functions, particularly in relation to calcium-phosphate-containing crystals. The manner in which a variety of synthetic and natural acidic macromolecules interact in vitro with crystals of octacalcium phosphate (OCP) has been studied. Interactions were assessed by examining changes in morphology of the crystals resulting from preferential interaction of the additive with some crystal faces and not others. Macromolecules rich in acidic amino acids, with or without polysaccharides, such as polyaspartate and mollusk shell proteins respectively, were shown to interact preferentially with rows of Ca ions exposed on the hydrated plate surface of OCP crystals. In contrast, the phosphorylated proteins, phosphophoryn and phosvitin, interacted specifically with the apatite-like motifs on the OCP side faces. BSP did not interact specifically with OCP, under the experimental conditions used. The observation that these classes of acidic macromolecules recognize different crystal faces should be taken into account when evaluating functions of acidic matrix macromolecules in mineralized tissues.

New method for discriminating between calcium stone formers and healthy individuals.

A new method for discriminating between the urine of potential calcium stone formers and healthy persons has been proposed, based on determination of the calcium binding capacity of urine (CBC) by titration of early morning urine with a calcium chloride solution. For this purpose a new PVC matrix calcium ion selectrode for measuring calcium ion concentration in whole urine was used. The selectrode has a disposable membrane which can easily be prepared and replaced in the laboratory. Plots of the calcium ion concentration versus the concentration of total added calcium were linear up to a point where precipitation of calcium salts commenced. The slopes of these titration lines were used as criteria for discrimination. Statistical evaluation showed good separation between the urine of healthy and stone forming donors. A 2-sample t test with unequal variances gave mean values of 0.31 for healthy urine (13 samples) and 0.64 for stone forming urine (26 samples). Individual 99% confidence intervals were 0.21-0.40 for the controls and 0.54-0.73 for the patients respectively. Discriminant analysis showed that from a group of treated patients with low Dl (13 samples), 3 were classified as stone formers and 10 were non-formers. Thus there was good correlation with the clinical situation and with the previously proposed Dl test.

Determination of the calcium binding capacity to discriminate between urines of calcium stone formers and healthy persons.

Calcium binding by undiluted fasting urine has been tested as a means of demonstrating the capacity of urine from control subjects and calcium stone patients to hold spontaneous precipitation of stone forming compounds. Preliminary data are promising with respect to the possibility that controls and patients can be separated.

Precipitation and Solubility of Calcium Hydrogenurate Hexahydrate.

Solid phases formed in the quaternary system: uric acid-calcium hydroxide -hydrochloric acid-water aged for 2 months at 310 K were studied to determine conditions for calcium hydrogenurate hexahydrate, Ca(C5H3N4O)2 · 6H2O precipitation. The precipitates were identified by chemical and thermogravimetric analyses, x-ray powder diffraction, infrared spectroscopy, light microscopy, and scanning electron microscopy. In the precipitation diagram the concentration region in which calcium hydrogenurate hexahydrate precipitated as a single solid phase was established. The solubility of calcium hydrogenurate hexahydrate was investigated in the pH range from 6.2 to 10.1 at different temperatures. The total soluble and ionic concentration of calcium (atomic absorption spectroscopy and Ca-selective electrode), total urate concentration (spectrophotometry), and pH were determined in equilibrated solutions. The data are presented in the form of tables and chemical potential diagrams. By using these data the thermodynamic solubility products of calcium hydrogenurate hexahydrate, Ks = a(Ca2+) · a2(C5H3N4O3-), were determined: [Formula: see text]The formation of calcium hydrogenurate hexahydrate crystals in urinary tract of patients with pathologically high concentrations of calcium and urates (hypercalciuria and hyperuricosiuria) is possible.

Precipitation of calcium phosphates from electrolyte solutions. V. The influence of citrate ions.

The influence of citrate ions on the precipitation of crystalline apatitic precipitates with low Ca/P molar ratios [octacalcium phosphate (OCP) and calcium-deficient apatites (DA) (system A)] and of the intercrystalline mixtures of calcium hydrogen phosphate dihydrate (DCPD) and DA (system B) was investigated. Samples were prepared by direct mixing of calcium chloride solutions (A, 6.10(-3) mol dm-3; B, 1.10(-1) mol dm-3) and sodium phosphate solutions (A, 6.10(-3) mol dm-3; B, 2.10(-2) mol dm-3) containing citrate (0-2.10(-3) mol dm-3) and preadjusted to pH 7.4. In the presence of citrate ions: (a) crystal growth of OCP and DA was slowed down; (b) habit modification of DCPD crystals occurred; and (c) equilibration in intercrystalline mixtures of DCPD and DA's was slowed down. All phenomena were caused by surface adsorption of negatively charged ions, most probably CaC6H5O7-, which is the prevalent calcium citrate species under the given experimental conditions. Habit modification of DCPD was induced by preferential adsorption at the (001) crystal plane.

Precipitation of calcium phosphates from electrolyte solutions. III. Radiometric studies of the kinetics of precipitation and aging of calcium phosphates.

Precipitation and precipitate transformation in the system sodium phosphate (pre-adjusted to pH 7.4)--calcium chloride (25 degrees) was studied by means of radiometric analysis using 45Ca and 32P as tracers. Changes in the pH and the total concentrations of calcium and phosphate were followed during solid phase formation and the data were used to calculate composition changes of the precipitates and their supernatants. In all investigated systems two-step precipitation was observed, the precursor being more basic than the secondary precipitate. The composition of the latter was mostly within the range of the composition of octacalcium phosphate. The course of further chemical changes was dependent on the pH established during secondary precipitation. The heterogeneous exchange of the radionuclides between the solid phase and their supernatant solutions was also followed as a function of time. The results indicate that recrystallization through the mother liquid accompanied by composition changes is the dominant mechanism of equilibrium of the solid phases.