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PubMed2002

Osteoblast-mediated mineral deposition in culture is dependent on surface microtopography.

Osteoblast phenotypic expression in monolayer culture depends on surface microtopography. Here we tested the hypothesis that mineralized bone nodule formation in response to osteotropic agents such as bone morphogenetic protein-2 (BMP-2) and dexamethasone is also influenced by surface microtopography. Fetal rat calvarial (FRC) cells were cultured on Ti implant materials (PT [pretreated], Ra = 0.6 microm; SLA [course grit blasted and acid etched], Ra = 4.0 microm; TPS [Ti plasma sprayed], Ra = 5.2 microm) in the presence of either BMP-2 (20 ng/ml) or 10(-8) M dexamethasone (Dex). At 14 days post-confluence, a homogenous layer of cells covered the surfaces, and stacks of cells that appeared to be nodules emerging from the culture surface were present in some areas on all three Ti surfaces. Cell proliferation decreased while alkaline phosphatase specific activity (ALPase) and nodule number generally increased with increasing surface roughness in both control and treated cultures. There was no difference in cell number between the control and Dex-treated cultures for a particular surface, but BMP-2 significantly reduced cell number compared with control or Dex-treated cultures. Treatment with Dex or BMP-2 further increased ALPase on all surfaces except for PT cultures with Dex. Dex had no effect on nodule area in cultures grown on PT or SLA disks, yet increased nodule number by more than 100% in cultures on PT disks. Though the effect of BMP-2 on nodule number was the same as Dex, BMP-2 increased nodule area on all surfaces except TPS, where area was decreased. Ca and P content of the cell layers in control cultures did not vary with surface roughness. However, cultures treated with Dex had increased Ca content on all surfaces, but the greatest increase was seen on SLA and TPS. BMP-2 increased Ca content in cultures on all surfaces, with the greatest increase on the PT surface. BMP-2 treatment increased P content on all surfaces, whereas Dex only increased P on rough surfaces. Of all cultures examined, the Ca/P weight ratio was 2:1 only on rough surfaces with BMP-2, indicating the presence of bone-like apatite. This was further validated by Fourier transform infrared (FTIR) imaging showing a close association between mineral and matrix on TPS and SLA surfaces with BMP-2-treated cells, and individual spectra indicated the presence of an apatitic mineral phase comparable to bone. In contrast, mineral on the smooth surface of BMP-2-treated cultures and on all surfaces where cultures were treated with Dex was not associated with the matrix and the spectra, not typical of bone apatite, implying dystrophic mineralization. This demonstrates that interactions between growth factor or hormone and surface microtopography can modulate bone cell differentiation and mineralization.

Alkaline Phosphatase↗

PubMed2003

Analysis of hydroxyapatite surface coverage by amelogenin nanospheres following the Langmuir model for protein adsorption.

The assembly of amelogenin protein into nanospheres is postulated to be a key factor in the stability of enamel extracellular matrix framework, which provides the scaffolding for the initial enamel apatite crystals to nucleate and grow. Adsorption isotherms were evaluated in order to investigate the nature of interactions of amelogenin nanospheres with hydroxyapaite crystals in solution, where their assembly status and particle size distribution are defined. We report that the adsorption isotherm of a recombinant mouse amelogenin (rM179) on synthetic hydroxyapatite crystals can be described using a Langmuir model indicating that amelogenin nanospheres adsorb onto the surface of apatite crystals as binding units with defined adsorption sites. The adsorption affinity and the maximum adsorption sites were 19.7 x 10(5) L/mol and 6.09 x 10(-7) mol/m2, respectively, with an r2 value of 0.99. Knowing the composition and particle size distribution of amelogenin nanospheres under the condition of adsorption experiments, we have calculated the number of nanospheres and the crystal surface area covered by each population of nanospheres at their maximum adsorption. It was found that total maximum binding covers 64% of the area unit. This observation supports the speculation that amelogenin binding onto apatite surface is selective and occurs only at certain sites.

Adsorption↗

PubMed2002

Rock phosphate solubilization and colonization of maize rhizosphere by wild and genetically modified strains of Penicillium rugulosum.

Maize root colonization and phosphate solubilizing activity of the fungus Penicillium rugulosum were assessed in a greenhouse trial using soil-plant microcosms. The bacterial gene hph conferring resistance to hygromicin B was introduced by electroporation in the wild-type strain IR-94MF1 of P. rugulosum and one transformant, w-T3, was selected. Maize plants were grown for 5 weeks in a P-poor soil and fertilized with a Florida apatite mineral, with Navay, an apatite rock deposit from Venezuela, or with simple superphosphate. Inoculation treatments included strain IR-94MF1, transformant w-T3 and two IR-94MF1 UV-induced mutants with enhanced (Mps++) or reduced (Mps-) in vitro mineral phosphate solubilizing activity. In the absence of P fertilization, inoculation with any P. rugulosum isolate significantly reduced the size of the total and P-solubilizing bacterial community present in maize rhizosphere. The bacterial community significantly increased in maize inoculated with IR-94MF1 and w-T3 when P was added as apatites Navay or Florida. All P. rugulosum strains were able to stimulate the growth of maize plants as indicated by 3.6 to 28.6% increases in dry matter yields. In the presence of rock phosphate, P uptake by maize plants inoculated with the two mutants Mps++ and Mps- was not always in agreement with their P-solubilizing phenotypes. Strain IR-94MF1 and transformant w-T3 increased P assimilation by the plants fertilized with Navay rock phosphate by 26 and 38%, respectively. In this treatment, w-T3 showed its highest significant maize rhizosphere colonization. With the simple superphosphate treatment, w-T3 increased P uptake in plants by 8% over the uninoculated control and also decreased significantly the community size of total bacteria, total fungi, and P-solubilizing fungi in the rhizosphere.

Biological Transport↗

PubMed2005

Fungal degradation of calcium-, lead- and silicon-bearing minerals.

The aim of this study was to examine nutritional influence on the ability of selected filamentous fungi to mediate biogenic weathering of the minerals, apatite, galena and obsidian in order to provide further understanding of the roles of fungi as biogeochemical agents, particularly in relation to the cycling of metals and associated elements found in minerals. The impact of three organic acid producing fungi (Aspergillus niger, Serpula himantioides and Trametes versicolor) on apatite, galena and obsidian was examined in the absence and presence of a carbon and energy source (glucose). Manifestation of fungal weathering included corrosion of mineral surfaces, modification of the mineral substrate through transformation into secondary minerals (i.e. crystal formation) and hyphal penetration of the mineral substrate. Physicochemical interactions of fungal metabolites, e.g. H+ and organic acids, with the minerals are thought to be the primary driving forces responsible. All experimental fungi were capable of mineral surface colonization in the absence and presence of glucose but corrosion of the mineral surface and secondary mineral formation were affected by glucose availability. Only S. himantioides and T. versicolor were able to corrode apatite in the absence of glucose but none of the fungi were capable of doing so with the other minerals. In addition, crystal formation with galena was entirely dependent on the availability of glucose. Penetration of the mineral substrates by fungal hyphae occurred but this did not follow any particular pattern. Although the presence of glucose in the media appeared to influence positively the mineral penetrating abilities of the fungi, the results obtained also showed that some geochemical change(s) might occur under nutrient-limited conditions. It was, however, unclear whether the hyphae actively penetrated the minerals or were growing into pre-existing pores or cracks.

Aspergillus niger↗

PubMed2005

New chemical treatment for bioactive titanium alloy with high corrosion resistance.

It was recently claimed that titanium metal and its alloys can bond to the living bone, without being coated by apatite (VPS coatings), but by being chemically and heat-treated. The bioactivity of treated titanium is of interest because of the opportunity to obtain orthopaedic or dental implants presenting, at the same time, high toughness, strength and fatigue resistance as well as bone-bonding ability. The bioactive behaviour of the treated implants is due to the presence of a modified surface, which, during soaking in body fluid, promotes the precipitation of apatite. The apatite formed is strongly bonded to the substrate and promotes living bone bonding. In this work were characterised samples of Ti-6Al-7Nb alloy with surfaces presenting a different chemical and mechanical state. The aim of the research was twofold. The first objective was to characterise chemically and heat-treated samples with different surface topography, in order to define the best conditions for osteogenic integration. The second aim was to assess the corrosion behaviour of the bioactive implants, because they expose a microporous and quite thin modified surface layer. No-treated and passivated samples, with a surface state closed to that nowadays used on implants, were used as reference. The surface structure, morphology, electrochemical behaviour and bioactivity of the different samples were assessed by means of XRD, SEM-EDS, anodic polarizations, open circuit measurements and in-vitro tests. Results evidence that it is possible to modify the surface of the Ti-6Al-7Nb alloy in order to obtain the formation of a bioactive layer and that the substrate roughness influences the characteristics of the surface layer formed. It was also evidenced that the as treated surfaces present inadequate corrosion behaviour, so a new two-step chemical treatment has been developed in order to obtain a bioactive material with good corrosion resistance.

Biocompatible Materials↗

PubMed2006

Organic-inorganic interaction and the growth mechanism of hydroxyapatite crystals in gelatin matrices between 37 and 80 degrees C.

The crystal development of hydroxyapatite[HAp] phase in gelatin[GEL] matrices was investigated in the temperature range 37 to 80 degrees C by using X-ray diffraction, scanning electron microscopy(SEM), thermoanalytical measurement(DT/TGA), Fourier-Transformed Infra-Red(FT-IR) spectroscopy, and transmission electron microscopy(TEM) with electron diffraction(ED). It was found that during the coprecipitation of apatite phase in GEL matrices and the next aging process the crystallites were formed and developed through the two reaction mechanisms of organic-inorganic interaction between apatite phase and GEL molecules, and thermodynamic reaction for the crystal growing. The analytical evidences showed that there was a definite competition between these two mechanisms with the reaction temperature. Below 50 degrees C the crystal development of HAp was greatly suppressed by the existence of the GEL molecules, indicating the heterogeneous nucleation by the supposed number of carboxyl groups in GEL. Above 50 degrees C the effective organic components as a template for the heterogeneous nucleation of apatite crystallites were greatly degraded and so more amount of inorganic ions could be favorably accredited on the preexisting crystallites in virtue of the limited nucleation chance, finally resulting in the crystal growth. At higher temperature pretty big HAp crystals were developed with the depletion of the organics to be bound with crystallites in the slurry solution. Presumably it is believed that the poisoning of the functional groups in GEL molecules was vigorously occurred in the phosphoric acid environment above approximately 50 degrees C.

Biocompatible Materials↗

PubMed1986

An investigation of the theoretical background for the stability of the calcium-phosphate salts and their mutual conversion in aqueous solutions.

A computer program for calculation of ion-activity products, in aqueous calcium-phosphate solutions and suspensions, was used to construct solubility diagrams which demonstrate the stability and instability fields of the calcium phosphates relative to various parameters. With increasing pH, the solubility of all calcium-phosphate salts decreased (with a solubility increase above pH 12); the most important factors in apatite solubility were pH, the fluoride concentration and the ion strength. It was inferred that apatite owes its instability below pH 5 to its increasing solubility relative to that of brushite. Calculation on amorphous tricalcium phosphate showed that this salt in the pH range above 5 was so soluble that the concurrent supersaturation with respect to either brushite or apatite would induce their formation causing the instability of the amorphous salt.

Calcium Phosphates↗

PubMed1994

Experiments on the initiation of calcium fluoride formation with reference to the solubility of dental enamel and brushite.

As calcium fluoride formation following topical application of fluoride may be responsible for at least some of the caries-reducing effect of fluoride, the concentration of fluoride necessary to induce its formation were examined. The aim was to determine the degree of supersaturation with respect to calcium fluoride necessary for inducing its spontaneous precipitation, with close reference to its possible formation and retention on dental hard tissue when topical fluoride solutions are used clinically. Powdered enamel or brushite were suspended for 4 h in aqueous solutions buffered at pH 7.2 and 5.0. After the equilibration, ion concentrations were determined and degrees of saturation with respect to apatite and brushite were calculated. In aqueous solutions at pH 7.2 and 5.0 with similar concentrations as those found in the equilibrated suspensions the fluoride concentration was adjusted to from 5 to 500 parts/10(6). After 2 h of gentle agitation the supernatant was analysed, the precipitate (if any) isolated and examined by X-ray diffraction. Initiation of spontaneous calcium fluoride formation required a calcium fluoride ion-activity product of 10(-7.6) or more, 300 parts/10(6) fluoride were necessary to initiate calcium fluoride formation in neutral solution saturated with enamel, and the increased solubility of enamel apatite at low pH allowed calcium fluoride formation from solutions with as low as 100 parts/10(6) fluoride. When phosphate was present in the solution a competing apatite formation could mask the calcium fluoride formation. In neutral solutions saturated with respect to brushite, spontaneous fluorapatite formation was initiated by 100 pats/10(6) fluoride. With 200 parts/10(6) fluoride a further competing formation of calcium fluoride occurred.

Calcium Fluoride↗

PubMed1995

Surface modifications of glass-reinforced hydroxyapatite composites.

Surface modifications of glass-reinforced hydroxyapatite composites immersed in a simulated physiological solution were studied using X-ray photoelectron spectroscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy. In the first stages of the apatite formation process, calcium and silicon ions were leached out from the surface of the composites. After 12 days of immersion, apatite crystals were detected on the surface indicating bioactive behaviour. Sodium ions attributed to sodium sulphate were also found in the apatite layer. Bound water on the surface also increased with immersion time.

Biocompatible Materials↗

PubMed1995

Transmission electron microscopic observation of heterogeneous fluoridated hydroxyapatites.

Two different types of heterogeneous fluoridated hydroxyapatites previously synthesized at 80 degrees C and pH 7.4, which might be composed of hydroxyapatite covered with fluorapatite (H-FAp) and fluorapatite covered with hydroxyapatite (F-HAp), were observed by high-resolution transmission electron microscopy. The transverse-sectional shapes of both apatites were clearly hexagonal. This confirmed that both apatites were highly crystallized. However, their shapes differed slightly from each other in detail:H-FAp was more slender and F-HAp was typically hexagonal, probably reflecting the shapes of the original core apatites, hydroxyapatite and fluorapatite. The portions thought to be synthesized with fluoride-free solution showed clear electron beam damage. The expanded lattice image observed at the edge of the F-HAp crystal coincided approximately with the a-axis dimension of 0.9443 nm obtained from the X-ray diffraction data of synthetic hydroxyapatite. After incubation in 0.5 mol l-1 acetate buffer solution (37 degrees C, pH 4.0) for one month, the fluorapatite-covered H-FAp maintained its sharp corners, while the hydroxyapatite-covered F-HAp showed rounded corners.

Hydroxyapatites↗

PubMed1993

Biodegradation and bioresorption of calcium phosphate ceramics.

The use of several calcium phosphate (Ca-P) materials for bone repair, augmentation, substitution and as coatings on metal implants has gained clinical acceptance in many dental and medical applications. These Ca-P materials may be of synthetic or natural origin, available in different physical forms (dense or macroporous, particles or blocks) and are used in bulk as coatings for metallic and non-metallic substrates or as components in composites, cements and bioactive glasses. Biodegradation or bioresorption of calcium phosphate materials implies cell-mediated degradation in vitro or in vivo. Cellular activity during biodegradation or bioresorption occurs in acid media; thus the factors affecting the solubility or the extent of dissolution (which in turn depends on the physico-chemical properties) of the Ca-P materials are important. Enrichment of the microenvironment due to the release of calcium and phosphate ions from the dissolving Ca-P materials affects the proliferation and activities of the cells. The increase in the concentrations of the calcium and phosphate ions promotes the formation of carbonate apatite which are similar to the bone apatite. The purpose of this invited paper is to discuss the processes of biodegradation or bioresorption of Ca-P materials in terms of the physico-chemical properties of these materials and the phenomena involved including the formation of carbonate apatite on the surfaces and in the vicinity of these materials. This phenomenon appears to be related to the bioactivity of the material and the ability of such materials to directly attach to bone and to form a uniquely strong material-bone interface.

Biodegradation, Environmental↗

PubMedDate not supplied

In vivo behavior of three different injectable hydraulic calcium phosphate cements.

Two dicalcium phosphate dihydrate (DCPD) hydraulic cements and one apatite hydraulic cement were implanted in epiphyseal and metaphyseal, cylindrical bone defects of sheep. The in vivo study was performed to assess the biocompatibility of the DCPD cements, using the apatite cement as control. After time periods of 2, 4 and 6 months the cement samples were clinically and histologically evaluated. Histomorphometrically the amount of new bone formation, fibrous tissue and the area of remaining cement were measured over time. In all specimens, no signs of inflammation were detectable either macroscopically or microscopically. All cements were replaced by different amounts of new bone. The two DCPD-cements showed the highest new bone formation and least cement remnants at 6 months, whereas the apatite was almost unchanged over all time periods.

Animals↗

PubMed2004

Processing and properties of porous poly(L-lactide)/bioactive glass composites.

Porous poly(L-lactide)/bioactive glass (PLLA/BG) composites were prepared by phase separation of polymer solutions containing bioactive glass particles (average particle size: 1.5 microm). The composite microstructures consist of a porous PLLA matrix with glass particles distributed homogeneously throughout. Large pores (>100 microm) are present in a network of smaller (<10 microm) interconnected pores. The porous microstructure of the composites was not significantly influenced by glass content (9 or 29 vol%), but silane pretreatment of the glass resulted in better glass incorporation in the matrix. Mechanical tests showed that an increase in glass content increased the elastic modulus of the composites, but decreased their tensile strength and break strain. Silane pretreatment enhanced the increase in modulus and prevented the decrease in tensile strength with increasing glass content. Composites soaked in simulated body fluid (SBF) at body temperature formed bone-like apatite inside and on their surfaces. The silane pretreatment of glass particles delayed the in vitro apatite formation. This bone-like apatite formation demonstrates the composites' potential for integration with bone.

Biocompatible Materials↗

PubMed2004

Fluor-hydroxyapatite sol-gel coating on titanium substrate for hard tissue implants.

Hydroxyapatite (HA) and fluor-hydroxyapatite (FHA) films were deposited on a titanium substrate using a sol-gel technique. Different concentrations of F- were incorporated into the apatite structure during the sol preparation. Typical apatite structures were obtained for all coatings after dipping and subsequent heat treatment at 500 degrees C. The films obtained were uniform and dense, with a thickness of approximately 5 microm. The dissolution rate of the coating layer decreased with increasing F- incorporation within the apatite structure, which demonstrates the possibility of tailoring the solubility by a functional gradient coating of HA and FHA. The cell proliferation rate on the coating layer decreased slightly with increasing F- incorporation. The alkaline phosphatase (ALP) activity of the cells on all the HA and FHA coated samples showed much higher expression levels compared to pure Ti. This confirmed the improved activity of cell functions on the substrates with the sol-gel coating treatment.

Adsorption↗

PubMed2004

Effect of sintered silicate-substituted hydroxyapatite on remodelling processes at the bone-implant interface.

Phase pure, sintered granules of hydroxyapatite (HA) and silicon-substituted hydroxyapatite (Si-HA) were implanted for 6 and 12 weeks in an ovine model. Samples containing the bone-implant interface were prepared for ultramicrotomy and transmission electron microscopy (TEM) using an anhydrous sample preparation procedure. The results demonstrate that the morphology of apatite deposits and the sequence of events at the interfaces of bone with pure HA and with Si-HA implants, were different. Organised collagen fibrils were first found at the bone/Si-HA interface after 6 weeks, whereas they were found only after 12 weeks around the pure HA implant. Many more nodular aggregates comprised of plate-like apatite crystallites were observed in the vicinity of Si-HA than around the pure HA after 12 weeks in vivo. These findings suggest that the incorporation of silicate ions into HA promotes processes of bone remodelling at the bone/HA interface. TEM observations suggested that the trabecular bone weaves over the Si-HA and that the collagen fibrils form a mechanical interlock with the Si-HA ceramic implants. High-resolution lattice imaging illustrated apatite crystallites contiguous with the Si-HA ceramic and revealed a direct relationship between the bone mineral and the Si-HA ceramic.

Animals↗

PubMed2004

A novel method to produce hydroxyapatite objects with interconnecting porosity that avoids sintering.

Porous objects of carbonated apatite were prepared by mixing polyvinyl alcohol fibres (PVA) and sodium chloride as porogens with nanocrystalline carbonated apatite powder. After cold isostatic pressing (CIP) and dissolving the porogens, the bioceramic showed an interconnecting porosity with pore diameters in the range of 250-400 microm. The method can be transposed to any material that is insoluble in water (like many polymers). Such objects are promising for bone regeneration because the interconnecting porosity in carbonated apatite provides a good environment for bone attachment and ingrowth.

Biocompatible Materials↗

PubMed2006

In vitro and in vivo reactivity of porous, electrosprayed calcium phosphate coatings.

The dissolution and/or precipitation behaviour of porous calcium phosphate (CaP) coatings, deposited using electrostatic spray deposition (ESD), was investigated (a) in vitro after soaking in simulated body fluid (SBF) for several time periods (2, 4, 8, and 12 weeks), and (b) in vivo after subcutaneous implantation of CaP-coated implants in the back of goats for identical time periods. Physical and chemical properties of coatings were characterized before and after in vitro/vivo testing by means of scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and energy dispersive spectroscopy. Moreover, part of the explants was prepared for light microscopical evaluation of the tissue response. In vitro, all apatitic ESD-coatings induced the formation of homogeneous and adherent CaP precipitation layers. Amorphous CaP, however, displayed a delayed precipitation of poorly adherent CaP layers, whereas heterogeneous calcification was observed on top of beta-TCP-coated substrates, indicating that beta-TCP and amorphous CaP coatings exhibit a poor ability of inducing calcification in SBF as compared to crystalline apatitic coatings. In vivo, no adverse tissue reactions (toxic effects/inflammatory cells) were observed using light microscopy, and all coatings became surrounded by a dense, fibrous tissue capsule after implantation. All ESD-coatings degraded gradually at a dissolution rate depending on the chemical phase (order of relative solubility: amorphous CaP approximately carbonate apatite>beta-TCP>carbonated hydroxyapatite), thereby enabling synthesis of CaP coatings with a tailored degradation rate.

Animals↗

PubMed2006

Biomimetic synthesis of calcium-deficient hydroxyapatite in a natural hydrogel.

A novel composite material consisting of calcium-deficient hydroxyapatite (CdHAP) biomimetically deposited in a bacterial cellulose hydrogel was synthesized and characterized. Cellulose produced by Gluconacetobacter hansenii was purified and sequentially incubated in solutions of calcium chloride followed by sodium phosphate dibasic. A substantial amount of apatite (50-90% of total dry weight) was homogeneously incorporated throughout the hydrogel after this treatment. X-ray diffractometry (XRD) showed that CdHAP crystallites had formed in the cellulose. XRD further demonstrated that the CdHAP was comprised of 10-50 nm anisotropic crystallites elongated in the c-axis, similar to natural bone apatite. Fourier transform infrared (FTIR) spectroscopy demonstrated that hydroxyl IR bands of the cellulose shifted to lower wave numbers indicating that a coordinate bond had possibly formed between the CdHAP and the cellulose hydroxyl groups. FTIR also suggested that the CdHAP had formed from an octacalcium phosphate precursor similar to physiological bone. Scanning electron microscopy (SEM) images confirmed that uniform approximately 1 microm spherical CdHAP particles comprised of nanosized crystallites with a lamellar morphology had formed in the cellulose. The synthesis of the composite mimics the natural biomineralization of bone indicating that bacterial cellulose can be used as a template for biomimetic apatite formation. This composite may have potential use as an orthopedic biomaterial.

Biomimetics↗