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Langmuir-Blodgett monolayer films of bacterial photosynthetic membranes and isolated reaction centers: preparation, spectrophotometric and electrochemical characterization.

The Langmuir-Blodgett (LB) film technique has been successfully applied to the construction of stable and photo-active films of chromatophore membranes and isolated reaction centers from two species of photosynthetic bacteria, Rhodobacter sphaeroides and Rhodopseudomonas viridis. LB films of these preparations were characterized at the air/water interface through compression isotherms and film stabilities. Films deposited on glass slides were analyzed by spectrophotometric and redox potentiometric techniques. The results obtained indicate that the in vivo properties of the photosynthetic apparatus in the deposited films are essentially unchanged. Furthermore, the pigments and redox cofactors in the films are highly oriented and offer a unique opportunity for structural and functional studies of the kind described in the accompanying paper (Biochim. Biophys. Acta 1057 (1991) 258-272).

Cytochrome c Group↗

Protein in sugar films and in glycerol/water as examined by infrared spectroscopy and by the fluorescence and phosphorescence of tryptophan.

Sugars are known to stabilize proteins. This study addresses questions of the nature of sugar and proteins incorporated in solid sugar films. Infrared (IR) and Raman spectroscopy was used to examine trehalose and sucrose films and glycerol/water solvent. Proteins and indole-containing compounds that are imbedded in the sugar films were studied by IR and optical (absorption, fluorescence, and phosphorescence) spectroscopy. Water is able to move in the sugar films in the temperature range of 20-300 K as suggested by IR absorption bands of HOH bending and OH stretching modes that shift continuously with temperature. In glycerol/water these bands reflect the glass transition at approximately 160 K. The fluorescence of N-acetyl-L-tryptophanamide and tryptophan of melittin, Ca-free parvalbumin, and staphylococcal nuclease in dry trehalose/sucrose films remains broad and red-shifted over a temperature excursion of 20-300 K. In contrast, the fluorescence of these compounds in glycerol/water solvent shift to the blue as temperature decreases. The fluorescence of the buried tryptophan in Ca-bound parvalbumin in either sugar film or glycerol/water remains blue-shifted and has vibronic resolution over the entire temperature range. The red shift for fluorescence of indole groups exposed to solvent in the sugars is consistent with the motion of water molecules around the excited-state molecule that occurs even at low temperature, although the possibility of static complex formation between the excited-state molecule and water or other factors is discussed. The phosphorescence yield for protein and model indole compounds is sensitive to the matrix glass transition. Phosphorescence emission spectra are resolved and shift little in different solvents or temperature, as predicted by the small dipole moment of the excited triplet state molecule. The conclusion is that the sugar film maintains the environment present at the glass formation temperature for surface Trp and amide groups over a wide temperature excursion. In glycerol/water these groups reflect local changes in the environment as temperature changes.

Biophysical Phenomena↗

Orientation and lateral mobility of cytochrome c on the surface of ultrathin lipid multilayer films.

We have previously shown that cytochrome c can be electrostatically bound to an ultrathin multilayer film having a negatively charged hydrophilic surface; furthermore, x-ray diffraction and absorption spectroscopy techniques indicated that the cytochrome c was bound to the surface of these ultrathin multilayer films as a molecular monolayer. The ultrathin fatty acid multilayers were formed on alkylated glass, using the Langmuir-Blodgett method. In this study, optical linear dichroism was used to determine the average orientation of the heme group within cytochrome c relative to the multilayer surface plane. The cytochrome c was either electrostatically or covalently bound to the surface of an ultrathin multilayer film. Horse heart cytochrome c was electrostatically bound to the hydrophilic surface of fatty acid multilayer films having an odd number of monolayers. Ultrathin multilayer films having an even number of monolayers would not bind cytochrome c, as expected for such hydrophobic surfaces. Yeast cytochrome c was covalently bound to the surface of a multilayer film having an even number of fatty acid monolayers plus a surface monolayer of thioethyl stearate. After washing extensively with buffer, the multilayer films with either electrostatically or covalently bound cytochrome c were analyzed for bound protein by optical absorption spectroscopy; the orientation of the cytochrome c heme was then investigated via optical linear dichroism. Polarized optical absorption spectra were measured from 450 to 600 nm at angles of 0 degrees, 30 degrees, and 45 degrees between the incident light beam and the normal to the surface plane of the multilayer. The dichroic ratio for the heme alpha-band at 550 nm as a function of incidence angle indicated that the heme of the electrostatically-bound monolayer of cytochrome c lies, on average, nearly parallel to the surface plane of the ultrathin multilayer. Similar results were obtained for the covalently-bound yeast cytochrome c. Furthermore, fluorescence recovery after photobleaching (FRAP) was used to characterize the lateral mobility of the electrostatically bound cytochrome c over the monolayer plane. The optical linear dichroism and these initial FRAP studies have indicated that cytochrome c electrostatically bound to a lipid surface maintains a well-defined orientation relative to the membrane surface while exhibiting measurable, but highly restricted, lateral motion in the plane of the surface.

Cytochrome c Group↗

Energy of adhesion of human T cells to adsorption layers of monoclonal antibodies measured by a film trapping technique.

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level.

Adsorption↗

Identification of diabetic retinopathy by stereoscopic digital imaging via teleophthalmology: a comparison to slide film.

BACKGROUND: Diabetic retinopathy is a leading cause of vision loss in North America. We compared mydriatic seven-field stereoscopic digital imaging to 100 ASA slide film photography for the identification of diabetic retinopathy via teleophthalmology. METHODS: Patients from a northern Alberta community with diabetes mellitus diagnosed by a physician were asked to participate in a teleophthalmology pilot project. Patients were enrolled at four different times between October 1999 and June 2000. Seven 30 degree fields of the retina were photographed with both slide film and digital imaging (resolution 2008 x 3040 pixels) through a dilated pupil. Slide film was developed and reviewed in a masked fashion by a retinal specialist. Digital images for each patient were transmitted by satellite to Edmonton and analysed a minimum of 2 months after the original slide film. Retinal abnormalities were graded with the use of the Early Treatment Diabetic Retinopathy Study extension of the modified Airlie House classification. We calculated the sensitivity and specificity of digital imaging for the identification of features of diabetic retinopathy as seen on slide film. Pearson's correlation coefficient was also calculated. RESULTS: A total of 121 patients (241 eyes), of whom 114 (94.2%) had non-insulin-dependent diabetes, participated in the study. The average duration of diabetes was 8.5 years. Of the 121 patients 57 (47.1%) had diabetic retinopathy, 12 (9.9%) had clinically significant macular edema and 2 (1.6%) had neovascularization. Pearson's correlation coefficient for the presence of retinopathy between slide film and stereoscopic digital imaging was 0.92 for microaneurysms, 0.80 for hemorrhages, 0.45 for intraretinal microvascular abnormalities, 0.32 for venous beading, 1.00 for neovascularization of the disc, 1.00 for neovascularization elsewhere in the retina and 0.97 for clinically significant macular edema (p < 0.001). The correlation between the two techniques for severe nonproliferative diabetic retinopathy (NPDR) was 0.86 and for high-risk proliferative diabetic retinopathy 1.00 (p < 0.001). INTERPRETATION: Stereoscopic digital imaging has a high level of correlation with slide film for the identification of most features of diabetic retinopathy, including microaneurysms, hemorrhage, severe NPDR, high-risk proliferative diabetic retinopathy and clinically significant macular edema.

Adolescent↗

X-ray diffraction and foam film investigations of PC head group interaction in water/ethanol mixtures.

The influence of ethanol on single phospholipid monolayers at the water/air interface and in foam films has been investigated. Grazing incidence X-ray diffraction investigations (GIXD) of Langmuir monolayers from 1,2-distearoyl-phosphatidylcholine (DSPC) spread on water subphases with different amounts of ethanol were performed. The thickness and free specific energy of formation of foam films stabilized by 1,2-dimyristoyl-phosphatidylcholine (DMPC) at different concentrations of ethanol in the film forming dispersions were measured. The GIXD investigations show that the tilt angle of the alkyl chains in the PC lipid monolayer decreases with increasing concentration of ethanol caused by a decrease of the diameter of the head groups. With increasing ethanol content of the solution also the thickness of the aqueous core of PC lipid foam films decreases. We assume that ethanol causes a decreasing probability for the formation of hydrogen bonds of water molecules to the PC head groups. The distinct difference between the effects of ethanol on lipid bilayers as described in the literature and on monolayers and foam films found in this study is discussed. Whereas PC monolayers at the water/air interface become unstable above 25 vol.% ethanol, the PC foam films are stable up to 50 vol.% ethanol. This is related to the decrease of the surface excess energy per lipid molecule by the interaction between the two film surfaces.

Ethanol↗

On the use of deuterated phospholipids for infrared spectroscopic studies of monomolecular films: a thermodynamic analysis of single and binary component phospholipid monolayers.

A thermodynamic study of monolayer mixing behavior was performed using binary lipid mixtures of DPPC + DOPG (7:1 mol:mol) as well as a mixture containing the phosphocholine acyl chain perdeuterated analog of DPPC, DPPC-d62 (i.e. 7:1 mol:mol DPPC-d62 + DOPG). An analysis was performed on these isotherms that calculated the Helmholtz excess free energy of mixing to determine potential thermodynamic differences in the fully protiated DPPC:DOPG monolayer versus the identical monolayer substituted with acyl chain perdeuterated DPPC (i.e. DPPC-d62). The main conclusions of this study are that: (1) the temperature-dependent thermodynamic phase behavior of DPPC-d62 differs substantially from that of DPPC, and these differences appear magnified when these molecules are studied as monomolecular films at the A/W interface as opposed to bulk phase systems; and (2) the calculated excess free energy of mixing values, deltaFxs(A), for the deuterium-containing 7:1 DPPC-d62:DOPG binary monolayer film show a type of near-ideal mixing behavior above 20 degrees C that is consistent with the 'squeezing-out' of the DOPG component in the binary mixture containing the DPPC-d62 component. This type of behavior does not occur in the graph of the excess free energy of mixing for the fully protiated 7:1 DPPC:DOPG binary monolayer film. The use of acyl chain perdeuterated DPPC (i.e. DPPC-d62) in binary monomolecular mixtures with other phospholipids produces a real and measurable difference on the thermodynamic properties of the monolayer when compared to the case of the fully protiated monomolecular film. In particular, the presence of DPPC-d62 in a 7:1 mol:mol DPPC-d62:DOPG binary monomolecular film may over-state the fluid nature of the monolayer at any given temperature, thereby leading to an over-estimate of the amount of material potentially 'squeezed out' of the monomolecular film. These results have implications for a commonly used IR spectroscopic method that relies on the incorporation of deuterium-labeled phospholipids into monolayers for vibrational spectroscopic analysis of individual components in a multicomponent monomolecular film.

1,2-Dipalmitoylphosphatidylcholine↗

The influence of previous films on screening mammographic interpretation and detection of breast carcinoma.

AIM: To establish if the availability of previous mammograms improves the detection rate of carcinomas and reduces supplementary examinations in the National Health Service Breast Screening Programme (NHSBSP). METHOD: Eight radiologists with varying experience but an interest in reporting mammograms reported 100 mammograms on two occasions, at least 1 month apart. In this selection of 100 mammograms we randomly inserted 12 abnormal films, with both benign and malignant changes present. These were chosen retrospectively with histological correlation of the abnormality. On the first occasion only the current films were reviewed, however, on the second occasion previous films were available for comparison. The films were viewed under standard viewing conditions, and the participants were asked to comment if they required further views or would refer the patient for either an ultrasound examination or surgical referral. RESULTS: Receiver operating curves (ROC) were constructed for the group's overall performance on each of the occasions and there was no difference in the curves. This indicates that the presence of previous films did not improve diagnostic accuracy. The presence of previous films did, however, lead to a significant reduction in the number of additional views and ultrasound examinations that would have been requested. The most significant reductions occurred in the group of normal mammograms. CONCLUSION: The presence of previous mammograms does not lead to an improvement in diagnostic accuracy. However, the presence of previous films led to significant reduction in additional examinations and ultrasound examinations.

Breast Neoplasms↗

Prediction of lubricating film thickness in UHMWPE hip joint replacements.

An elastohydrodynamic lubrication model developed for a ball-in-socket configuration in a previous studies by the present authors (Jalali-Vahid et al., Thinning films and tribological interfaces, 26th Leeds-Lyon Symposium on Tribology, 2000, pp. 329-339) was applied to analyse the lubrication problem of a typical artificial hip joint replacement, consisting of an ultra-high molecular weight polyethylene (UHMWPE) acetabular cup against a metallic or ceramic femoral head. The cup was assumed to be stationary whilst the ball was assumed to rotate at a steady angular velocity and under a constant load. A wide range of main design parameters were considered. It has been found that the predicted lubricating film thickness increases with a decrease in the radial clearance, an increase in the femoral head radius, an increase in UHMWPE thickness and a decrease in UHMWPE modulus. However, the predicted lubricating film thicknesses are not found to be sufficiently large in relation to the surface roughness of the cup and head to indicate separation of the two articulating surfaces. It should also be noted that if the design features are unable to secure full fluid film lubrication, it may be preferable to select them for minimum wear rather than maximum film thickness. For example, an increase in head radius will enhance the film thickness, but it will also increase the sliding distance and hence wear in mixed or boundary lubrication conditions. Furthermore, it is pointed out that an increase in the predicted lubricant film thickness is usually associated with an increase in the contact area, and this may cause lubricant starvation and stress concentration at the edge of the cup, and adversely affect the tribological performance of the implant. The effect of running-in process on the lubrication in UHMWPE hip joint replacements is also discussed.

Biocompatible Materials↗

Film drainage between two surfactant-coated drops colliding at constant approach velocity.

The drainage of the intervening continuous phase film between two drops approaching each other at constant velocity under the influence of insoluble surfactant is investigated. The mathematical model to be solved is a coupled pair of fourth-order nonlinear partial differential equations which arise from the relationships governing the evolution of the film thickness and the surfactant interfacial concentration in the lubrication approximation. We adopt a simplified approach which uses lubrication theory to describe the flow within the drop, marking a departure from the conventional framework in which Stokes flow is assumed. When the model is solved numerically together with the relevant initial and boundary conditions, the results obtained are compared with those found in the literature using the "boundary integral" method to solve for the flow in the drop phase. The close agreement between the results inspires confidence in the predictions of the simplified approach adopted. The analysis on the effect of insoluble surfactant indicates that its presence retards the drainage of the film: The fully immobile interface limit is recovered even in the presence of a small amount of surfactant above a critical concentration; film rupture is either prolonged or prevented. The retardation of the film was attributed to gradients of interfacial tension which gave rise to the Marangoni effect. A study of the influence of various system parameters on the drainage dynamics was conducted and three regimes of drainage and possible rupture were identified depending on the relative magnitudes of the drop approach velocity and the van der Waals interaction force: Nose rupture, rim rupture, and film immobilization and flattening. Finally, the possibility of forming secondary droplets by encapsulating the continuous phase film into the coalesced drop at rupture was examined and quantified in light of these regimes.

Journal Article↗

Void closure and interdiffusion processes during latex film formation from surfactant-free polystyrene particles: a fluorescence study.

This study reports a steady state fluorescence (SSF) technique for studying film formation from surfactant-free polystyrene (PS) latex particles. The latex films were prepared from pyrene (P)-labeled PS particles at room temperature and annealed at elevated temperatures for 5-, 10-, 20-, and 30-min time intervals above the glass transition (T(g)) temperature of polystyrene. During the annealing processes, the transparency of the film changed considerably. Scattered light (I(sc)) and fluorescence intensity (I(0P)) from P were measured after each annealing step to monitor the stages of film formation. Evolution of transparency of latex films were monitored using photon transmission intensity, I(tr). Scanning electron microscopy (SEM) was used to detect variation in the physical structure of annealed films. Onset temperature for film formation, T(0), void closure, T(v), and healing temperatures, T(h), were determined and corresponding activation energies were measured. Void closure and interdiffusion stages were modeled and related activation energies were determined.

Journal Article↗

Assembly process of 2D protein arrays in wetting films.

We were successful in developing a technique to form protein array developed directly on solid surfaces. This promising array formation revealed two new concepts of the ordering mechanism: the lifetime of a secondary minimum and secondary films. These concepts are not limited to our film formation technique. Any electrolytic thin film on a solid surfaces or any free film may contain a secondary film. If the secondary film can be retained in an electrolytic thin film for a sufficient period, a large single domain of small colloidal particles, such as proteins, fine metal particles, fine semi-conductive particles, etc., can be produced by an Alder-type transition.

Air↗

Diagnostic usefulness of post-void film in intravenous urogram.

A survey was sent to urologists to assess their subjective evaluation of the post-void film in the intravenous urogram (IVU). Most of the urologists believed that the post-void film should be a routine part of all IVUs, particularly in men over forty years of age, and estimated that the film contributed useful information in 30 percent of cases. One hundred fifty IVUs were reviewed to assess the actual value of the post-void film in 119 different patients. In these patients, the postvoid film was seen to give unique information not available by history or films of the filled bladder in only 3 percent of the cases. Therefore, we conclude the post-void film should not be a routine part of every IVU, and should be obtained on an as-needed basis only.

Adult↗

Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing.

Chemically-crosslinked glycosaminoglycan (GAG) hydrogel films were prepared and evaluated as bio-interactive wound dressings. Hyaluronan (HA) and chondroitin sulfate (CS) were first converted to the adipic dihydrazide derivatives and then crosslinked with poly(ethylene glycol) propiondialdehyde to give a polymer network. The crosslinking occurred at neutral pH in minutes at room temperature to give clear, soft hydrogels. After gelation, a solvent-casting method was used to obtain a GAG hydrogel film. A mouse model was used to evaluate the efficacy of these GAG films in facilitating wound healing. Full-thickness wounds were created on the dorsal side of Balb/c mice and were dressed with a GAG film plus Tegaderm' or TegadermT' alone. A significant increase in re-epithelialization was observed on day 5 (p < 0.001) and day 7 (p < 0.05) for wounds treated with a GAG film plus Tegaderm versus those treated with Tegaderm alone. While no significant differences in wound contraction or inflammatory response were found, wounds treated with either HA or CS films showed more fibro-vascular tissue by day 10. The GAG hydrogel films provide a highly hydrated, peri-cellular environment in which assembly of other matrix components. presentation of growth and differentiation factors, and cell migration can readily occur.

Animals↗

In vitro calcium phosphate growth over surface modified PMMA film.

In vitro nucleation of calcium phosphate phase was studied over functionalized polymethyl methacrylate (PMMA) films using Fourier transform infrared spectroscopy, electron spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. PMMA films were prepared by dissolving commercial grade pellets in chloroform and cast into thin sheets. The films were immersed in a methanol solution of sodium hydroxide before treating with 1.5% solution of adenosine triphosphate (ATP) at a pH of 5.2 for 24 h. ATP treated films were then soaked in saturated lime solution for 4 days to initiate formation of calcium phosphate precursor phase over their surface. The above films immersed in simulated body fluid solution (1.5 x SBF) for more than 5 days led to the nucleation of apatitic calcium phosphate phase all over the film surface. The ATP coupled film not subjected to lime treatment did not show calcium phosphate nucleation behaviour upon immersion in SBF solution. The Ca/P ratio of the calcium phosphate phase increase with increase in soaking time in SBF solution.

Calcium Phosphates↗

Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification.

Endothelial cell seeding constitutes an appreciated method to improve blood compatibility of small-diameter vascular grafts. In this study, we report the development of a simple innovative technique based on multilayered polyelectrolyte films as cell adhesive substrates. Polyelectrolyte multilayered films ending by poly(sodium-4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) or poly(L-glutamic acid)/poly(D-lysine) (PGA/PDL) could enhance cell adhesion by modification of the physico-chemical properties of the surface. The biological responses of human umbilical vein endothelial cells seeded on the polyelectrolyte multilayer films, on PDL or PAH monolayers, and on control surfaces, were evaluated in terms of initial attachment, growth, cellular metabolic activity, endothelial phenotype, and adhesion. The results showed that polyelectrolyte multilayers neither induce cytotoxic effects nor alter the phenotype of the endothelial cells. The polyelectrolyte multilayered films enhanced initial cell attachment as compared to the polyelectrolyte monolayer. Cell growth observed on the films was similar to that on TCPS. Among the different coating tested, the film ending by PSS/PAH exhibited an excellent cellular biocompatibility and appeared to be the most interesting surface in terms of cellular adhesion and growth. Such films could be used to cover hydrophobic (cell resistant) substrates in order to promote cell colonization, thereby constituting an excellent material for endothelial cell seeding.

Biocompatible Materials↗

In vitro and in vivo degradation of films of chitin and its deacetylated derivatives.

Chitin was deacetylated to various extents with NaOH to obtain partially and thoroughly deacetylated chitins. The specimens used in this study were deacetylated by 0 (chitin), 68.8, 73.3, 84.0, 90.1 and 100 mol% (chitosan). Films with a thickness of 150 microns were prepared from these specimens by the solution casting method. The equilibrated water contents of the films were 52.4 (chitin), 73.8 (68.8 mol%), 64.2 (73.3 mol%), 61.8 (84.0 mol%), 57.8 (90.1 mol%) and 49.7 wt% (chitosan), while the tensile strengths of the water-swollen films were 244 (chitin), 197 (68.8 mol%), 232 (73.3 mol%), 320 (84.0 mol%), 293 (90.1 mol%) and 433 g mm-2 (chitosan). The maximum water content and the minimum tensile strength observed for a specimen deacetylated between 0 and 68.8 mol% may be ascribed to the lowered crystallinity by deacetylation of chitin, since both chitin and chitosan are crystalline polymers. Unlike their physical properties, in vitro and in vivo degradations of these films occurred less rapidly without passing a maximum or minimum, as their degree of deacetylation became higher. The in vitro degradation was carried out by immersing the films in buffered aqueous solution of pH 7 containing lysozyme at 37 degrees C, while the in vivo degradation was studied by subcutaneously implanting the films in the back of rats. It was found that the rate of in vivo biodegradation was very high for chitin and 68.8 mol% deacetylated chitin, compared with that for the 73.3 mol% deacetylated chitin. The films which were more than 73.3 mol% deacetylated showed slower biodegradation. Interestingly, the tissue reaction towards highly deacetylated derivatives including chitosan was very mild, although they had cationic primary amines in the molecule.

Acetylation↗

Porous calcium phosphate coating over phosphorylated chitosan film by a biomimetic method.

A porous calcium phosphate coating deposited on chitosan films was studied using scanning electron microscopy, energy-dispersive X-ray analysis, micro-Fourier transform infrared spectroscopy (micro-FTIR) and thin-film X-ray diffractometry (XRD). Chitosan films were first prepared by dissolving chitosan powder in dilute acetic acid and drying in a flat petri dish. The films were phosphorylated using urea and H3PO4 with the P content being 0.1-0.2 wt%. Phosphorylated films soaked in saturated Ca(OH)2 solution for 8 days led to the formation of a calcium phosphate precursor phase over the entire surface. This precursor phase stimulated the growth of a porous coating of calcium-deficient hydroxy apatite when immersed in 1.5 x SBF for more than 20 days. Phosphorylated films not treated with Ca(OH)2 did not show any calcium phosphate growth upon immersion in SBF solution. The precursor phase is thought to be octacalcium phosphate, which nucleates a HAP phase during SBF treatment. Initially, this treatment in SBF results in the formation of a single-layer calcium phosphate particles over the film surface. As immersion time in SBF increases, further nucleation and growth produce a porous HAP coating. The Ca/P ratio of the HAP coating is a function of SBF immersion time.

Absorptiometry, Photon↗