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Coating of an apatite layer on polyamide films containing sulfonic groups by a biomimetic process.

Coating organic polymers with hydroxyapatite is an attractive method for the development of materials for medical applications, as it allows hydroxyapatite to show its unique biological properties such as its ability for bone bonding and protein adsorption. The biomimetic process focuses attention on fabricating such hydroxyapatite-polymer hybrids, where bone-like apatite is deposited on an organic polymer surface in solutions mimicking physiological conditions. In this process, a bone-like apatite layer can be coated onto organic substrates either by using a simulated body fluid (SBF), which has ion concentrations nearly equal to those of human extracellular fluid, or by using fluids that are supersaturated with respect to apatite at ambient conditions. We previously reported that apatite was deposited on polyamide films containing carboxyl groups in a solution mimicking body fluid, when they were incorporated with calcium salts. In the present study, to find an alternative functional group effective in apatite formation, we examined the apatite-forming ability of polyamide films containing sulfonic groups in the same solution. It was found that the polyamide film containing sulfonic groups could deposit apatite on its surface in the solution when the film was incorporated with calcium salts. These results show that the sulfonic group also acts as a functional group, and is as effective for apatite deposition in the body environment as the carboxyl group.

Biocompatible Materials↗

A new glance at ruthenium sorption mechanism on hydroxy, carbonate, and fluor apatites: Analytical and structural studies.

The sorption mechanism of Ru3+ ions on hydroxy (HAp), carbonate (CO3HAp), and fluor apatites (FAp) has been studied in detail. Ru apatites were obtained by reaction of the apatites with RuCl3 in aqueous solution. The structure and composition of the ruthenium-modified apatites were studied by several techniques: elemental analysis, XRD, EXAFS, IR, NMR, SEM-EDS, TEM, and thermal analysis. The amount of Ru in the modified apatite varies from 7.8 to 10.5 wt% and is not related to the initial composition or the specific surface area of the apatite. The different characterization techniques show that in the Ru-modified apatites Ru is surrounded by six oxygen atoms and do not contain any chlorine. For Ru-HAp and Ru-CO3HAp the new phase is amorphous whereas it is crystalline for FAp. The catalytic oxidation ability is higher for Ru-HAp and Ru-CO3HAp compared to Ru-FAp apatite in the oxidation of benzylic alcohol.

Journal Article↗

Composition and structure of apatite formed on organic polymer in simulated body fluid with a high content of carbonate ion.

Apatite layer was formed on polyethyleneterephthalate (PET) substrate by the following biomimetic process. The PET substrate was placed on granular particles of a CaO, SiO2-based glass in simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma to form apatite nuclei on their surfaces. The apatite nuclei was then grown into a continuous layer by subsequently soaking the substrate in SBF under air or CO2 atmosphere in which CO2 partial pressure in the ambient was adjusted to 14.8 kPa to increase the content of carbonate ion to a level nearly equal to that of blood plasma. The increase in the content of carbonate ions in SBF changed the Ca/P atomic ratio of the apatite from 1.51 to 1.63, content of CO(3)2- ions from 2.64 to 4.56 wt %, and lattice constants a from 94.32 to 94.23 nm and c from 68.70 to 68.83 nm, respectively. The Ca/P ratio and lattice constants of the apatite formed in SBF under CO2 atmosphere were approximately identical to those of bone apatite, i.e. Ca/P atomic ratio 1.65, content of CO(3)2- ion 5.80 wt % and lattice constants a 94.20 and c 68.80 nm. This indicates that an apatite with composition and structure nearly identical to those of bone apatite can be produced in SBF by adjusting its ion concentrations including the content of carbonate ions to be equal to those of blood plasma.

Journal Article↗

Enhancement of bonding strength by graded structure at interface between apatite layer and bioactive tantalum metal.

Tantalum metal is a candidate for use as an implant material in high load-bearing bony defects, due to its attractive features such as high fracture toughness and high workability. This metal, however, does not have bone-bonding ability, i.e. bioactivity, and therefore the development of bioactive tantalum metal is highly desirable. It is known that the essential prerequisite for an artificial material to show bioactivity is to form a bonelike apatite layer on its surface in the body environment. The same type of apatite layer is formed in a simulated body fluid (SBF) with inorganic ion concentrations nearly equal to those of human blood plasma. The present authors previously showed that the apatite formation on tantalum metal in SBF was remarkably accelerated by treatment with 0.5 M-NaOH aqueous solution and subsequent firing at 300 degrees C, while untreated tantalum metal spontaneously formed the same apatite after a long soaking period. In the present study, the bonding strength of the apatite layer to the substrate was quantitatively evaluated in comparison with that to the untreated tantalum metal. Adhesive strength was measured as an estimation of bonding strength, and the surface microstructure of both the substrates was characterized in order to discuss the difference in the bonding strength in terms of surface structure. The apatite layer formed on the NaOH- and heat-treated tantalum metal shows higher adhesive strength than that formed on the untreated metal. The amorphous sodium tantalate layer formed on the tantalum metal by NaOH and heat treatments, has a smooth graded structure where its concentration gradually changes from the surface into the interior metal. Smooth graded structure with complex of apatite is constructed after soaking in SBF. The higher bonding strength of the apatite layer formed on the treated metal is attributed to its smooth graded structure.

Journal Article↗

Apatite deposition on polyamide films containing carboxyl group in a biomimetic solution.

The development of organic-inorganic hybrids composed of hydroxyapatite and organic polymers is attractive because of their novelty in being materials that show a bone-bonding ability, i.e. bioactivity, and because they have mechanical properties similar to those of natural bone. The biomimetic process has received much attention for fabricating such a hybrid, where bone-like apatite is deposited under ambient conditions on polymer substrates in a simulated body fluid (SBF) having ion concentrations nearly equal to those of human extracellular fluid or related solutions. It has been shown that the carboxyl group is effective for inducing heterogeneous nucleation of apatite in the body. In the present study, apatite deposition on polyamide films containing various numbers of carboxyl groups was investigated in 1.5 SBF, which had ion concentrations 1.5 times those of a normal SBF. The effect of incorporation of calcium chloride on the formation of apatite was examined. Polyamide films containing or=40 mass % CaCl(2) formed apatite on their surfaces in 1.5 SBF. The ability of the modified film to form an apatite layer increased, and the adhesion of the apatite layer bonded to the film improved, with increasing carboxyl group content. It is concluded that novel apatite-polyamide hybrids can be prepared by a biomimetic process.

Journal Article↗

Bone bonding ability of an apatite-coated polymer produced using a biomimetic method: a mechanical and histological study in vivo.

A 20-microns thick apatite layer was coated onto polyethersulfone (PES) rectangular plates by soaking them in simulated body fluid containing CaO-SiO2 based glass powder. Coated and uncoated PES plates (10 x 15 x 1.5 mm) were implanted in the tibiae of rabbits, which were sacrificed 8, 16, and 30 weeks thereafter, and the samples were examined histologically using contact microradiography (CMR), Giemsa surface staining, and a scanning electron microscope connected to an electron probe microanalyzer (SEM-EPMA). The tensile failure loads at the bone/implant interfaces were determined using the detaching test. The histological examinations showed excellent bone apposition on coated PES and the sign of degradation of the apatite layer at remodeling lacunae. The apatite layer underwent complete resorption and was replaced by bone in most areas of the bone/implant interface after 30 weeks. Bone did not bond directly to uncoated PES after each follow-up period. The failure loads between bone and coated PES at 8, 16, and 30 weeks after implantation were 1.7 +/- 0.35, 2.36 +/- 0.53, and 1.45 +/- 0.48 kg, respectively. Those between bone and uncoated PES were nearly 0 kg at each postimplantation period. Failure during the detaching test occurred at the bone/apatite interface or near it after 8 weeks. After 16 weeks, it usually occurred at the apatite/ PES interface or near it, and occasionally in the middle of the apatite layer. The apatite layer was hardly detected at the failured interface after 30 weeks. In this study, an apatite-coated PES produced using a biomimetic method was demonstrated to bond directly to bone without any intervening soft tissue, which indicates that this material possesses excellent bioactivity.

Animals↗

Quasi-biological apatite film induced by titanium in a simulated body fluid.

Commercially pure titanium (c.p.Ti) is capable of inducing the formation of a carbonated apatite onto its surface in a simulated body fluid (SBF) comprised of calcium ions, phosphate ions, and other inorganic species present in the body fluid. In addition to the incorporation of carbonate ions, such formed apatite has other important characteristics of the bone mineral phase, such as a small crystal size and ionic substitution by Mg2+ and Cl-. Thus, we call this apatite a quasi-biological apatite. The formation of the quasi-biological apatite is proposed to be related to TiOH groups that develop on the titanium surface through interaction with the SBF. The results suggest that titanium implants may be activated such that they can form a strong bond with bone tissue through the in vivo formation of apatite. Since the solution can reach any open space, the process discussed in this study is very suitable for coating porous titanium implants with a quasi-biological apatite film.

Apatites↗

Qualitative assessment of natural apatite in vitro and in vivo.

Among the natural and synthetic materials investigated as bone graft substitutes, much interest has been focused on natural apatite obtained from low temperature heat-deproteinated compact bone. Previous research demonstrates that, when treated at a temperature below 500 degrees C, this material maintains its characteristic ultrastructural features, with a high surface/volume ratio, while as an implant material, it offers the host tissue a large surface of interaction. In vitro and in vivo tests showed that natural apatite is well tolerated and is a good osteoconducing material. The present in vivo study in rabbits was carried out to first investigate the behavior and capacity of natural apatite implants to stimulate bone ingrowth, and then to analyze the cells located at the bone/material interface. Synthetic hydroxyapatite was used as a control material. In a parallel in vitro study, we investigated the activity of differentiated osteoblasts and periosteal cells obtained from rats and new-born rabbits, incubated with natural apatite and synthetic hydroxyapatite. The in vivo study showed that natural apatite allows osteoblasts to form new bone tissue, adhering to the implant with ingrowth into the implant structure. In the presence of synthetic hydroxyapatite, a less pronounced osteoblastic activity was observed. In agreement with these observations, the in vitro study showed that natural apatite is more effective in attracting cells, favoring their proliferation and stimulating alkaline phosphatase activity. These findings suggest that natural apatite is more suitable for bone filling or bone regeneration than synthetic hydroxyapatite.

Animals↗

Poly(DL-lactic-co-glycolic acid) sponge hybridized with collagen microsponges and deposited apatite particulates.

A novel three-dimensional porous scaffold has been developed for bone tissue engineering by hybridizing synthetic poly(DL-lactic-co-glycolic acid) (PLGA), naturally derived collagen, and inorganic apatite. First, a porous PLGA sponge was prepared. Then, collagen microsponges were formed in the pores of the PLGA sponge. Finally, apatite particulates were deposited on the surfaces of the collagen microsponges in the pores of PLGA sponge. The PLGA-collagen sponge served as a template for apatite deposition, and the deposition was accomplished by alternate immersion of PLGA-collagen sponge in CaCl(2) and Na(2)HPO(4) aqueous solutions and centrifugation. The deposited particulates were small and scarce after one cycle of alternate immersion. Their number and size increased with the number of alternate immersion cycles. The surfaces of collagen microsponges were completely covered with apatite after three cycles of alternate immersion. The porosity of the hybrid sponge decreased gradually as the number of alternate immersion increased. Energy-dispersive spectroscopy analysis and X-ray diffraction spectra showed that the calcium-to-phosphorus molar ratio of the deposited particulates and the level of crystallinity increased with the number of alternate immersion cycles, and became almost the same as that of hydroxyapatite after four cycles of alternate immersion. The deposition process was controllable. Use of the PLGA sponge as a mechanical skeleton facilitated formation of the PLGA-collagen-apatite hybrid sponge into desired shapes and collagen microsponges facilitated the uniform deposition of apatite particulates throughout the sponge. The PLGA-collagen-apatite hybrid sponge would serve as a useful three-dimensional porous scaffold for bone tissue engineering.

Apatites↗

Apatite formation on the surface of Ceravital-type glass-ceramic in the body.

Previous studies on surface structural changes in vitro as well as in vivo of bioactive A-W-type glass-ceramics and Bio-glass-type glasses showed that the essential condition for glasses and glass-ceramics to bond to living bone is formation of a bonelike apatite layer on their surfaces in the body. Gross et al., however, had explained the bone-bonding mechanism of Ceravital-type apatite-containing glass-ceramic without mentioning formation of the surface apatite layer. In the present study, apatite formation on the surface of one of Ceravital-type glass-ceramics was investigated in vitro as well as in vivo. An apatite-containing glass-ceramic of the composition Na2O 5, CaO 33, SiO2 46, Ca(PO3)2 16 wt%, which was named KGS by Gross et al., was soaked in an acellular simulated body fluid which had ion concentrations almost equal to those of the human blood plasma. The same kind of glass-ceramic was implanted into a rabbit tibia. Thin-film x-ray diffraction, Fourier transform infrared reflection spectroscopy, and scanning electron microscopic observation of the surfaces of the specimens soaked in the simulated body fluid showed that Ceravital-type glass-ceramic also forms a layer of carbonate-containing hydroxyapatite of small crystallites and/or a defective structure on its surface in the fluid. Electron probe x-ray microanalysis of the interface between the glass-ceramic and the surrounding bone showed that a thin layer rich in Ca and P is present at the interface. These findings indicated that Ceravital-type glass-ceramics also form the bonelike apatite layer on its surface in the body and bond to living bone through the apatite layer.

Animals↗

Macroporous and nanofibrous polymer scaffolds and polymer/bone-like apatite composite scaffolds generated by sugar spheres.

Scaffolds are crucial to tissue engineering/regeneration. In this work, a technique combining a unique phase-separation process with a novel sugar sphere template leaching process has been developed to produce three-dimensional scaffolds. The resulting scaffolds possess high porosities, well connected macropores, and nanofibrous pore walls. The technique advantageously controls macropore shape and size by sugar spheres, interpore opening size by assembly conditions (time and temperature of heat treatment), and pore wall morphology by phase-separation parameters. The bioactivity of a macroporous and nanofibrous poly(L-lactic acid) (PLLA) scaffold was demonstrated by the bone-like apatite deposition throughout the scaffold in a simulated body fluid (SBF). Preincorporation of nanosized hydroxyapatite eliminated the induction period and facilitated the apatite growth in the SBF. Interestingly, the apatite growth primarily occurred on the surface of the pores (internal and external) but not the interior of the nanofibrous network away from the pore surface. It was also noticed that the macropore size did not affect the apatite growth rate, while the interpore opening size did. The compressive modulus also increased substantially when a continuous apatite layer was formed on the pore walls of the scaffold. The resulting composite scaffold mimics natural bone matrix with the combination of an organic phase (a polymer such as PLLA) and an inorganic apatite phase. The demonstrated bioactivity of apatite layer, together with well-controlled macroporous and nanofibrous structures, makes the novel nanocomposite scaffolds desirable for bone tissue engineering.

Apatites↗

Surface functional group dependent apatite formation on bacterial cellulose microfibrils network in a simulated body fluid.

The apatite forming ability of biopolymer bacterial cellulose (BC) has been investigated by soaking different BC specimens in a simulated body fluid (1.5 SBF) under physiological conditions, at 37 degrees C and pH 7.4, mimicking the natural process of apatite formation. From ATR-FTIR spectra and ICP-AES analysis, the crystalline phase nucleated on the BC microfibrils surface was calcium deficient carbonated apatite through initial formation of octacalcium phosphate (OCP) or OCP like calcium phosphate phase regardless of the substrates. Morphology of the deposits from SEM, FE-SEM, and TEM observations revealed the fine structure of thin film plates uniting together to form apatite globules of various size (from <1 mum to 3 mum) with respect to the substrates. Surface modification by TEMPO (2,2,6,6-tetramethylpyperidine-1-oxyl)-mediated oxidation, which can readily form active carboxyl functional groups upon selective oxidation of primary hydroxyl groups on the surface of BC microfibrils, enhanced the rate of apatite nucleation. Ion exchanged treatment with calcium chloride solution after TEMPO-mediated oxidation was found to be remarkably different from other BC substrates with the highest deposit weight and the smallest apatite globules size. The role of BC substrates to induce mineralization rate differs according to the nature of the BC substrates, which strongly influences the growth behavior of the apatite crystals.

Acetobacter↗

Formation of bone-like apatite enhanced by hydrolysis of octacalcium phosphate crystals deposited in collagen matrix.

It has been shown that granules of synthetic octacalcium phosphate (OCP) or the composites with collagen are capable of enhancing bone regeneration, accompanied by a gradual conversion from OCP to apatite with time. The present study was designed to investigate whether formation of bone-like apatite can be accelerated by OCP deposited throughout collagen matrix (OCP collagen complex, OCC) immersed in simulated body fluid (SBF). The formation of bone-like apatite has been suggested to be essential to induce osteoconductivity of various substrates. The formation of OCP in collagen solution was investigated in calcium or phosphate ions in the range between 22.5 and 142.5 mM and pH 6.26 and 8.56. X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy (SEM) showed that condition to nucleate OCP was limited to that of a solution with Ca/P 0.43 around pH 7.16 in the presence of collagen. OCP was shown to be formed throughout the collagen matrix by SEM observation. The immersion of OCC in SBF up to 10 days enhanced apatite crystal deposition, probably through OCP-apatite conversion: the apatite formation in OCC took place within only 1 day. The present study indicated that the existence of OCP deposited throughout the collagen matrix promotes bone-like apatite formation under physiological condition.

Animals↗

Types of "H2O" in human enamel and in precipitated apatites.

Types of "H2O" in human enamel and in precipitated apatites are characterized using X-ray diffraction, infrared (IR) absorption spectroscopic and thermogravimetric analyses. Changes in lattice parameters (principally in the a-axis dimensions) and in the character of the IR absorption bands are correlated with weight losses at pyrolysis temperatures of 100 degrees to 400 degrees C and with effect of rehydration and reignition of previously ignited samples. This study demonstrated that the loss of "H2O" below 200 degrees C is reversible and causes no significant change in the lattice parameter of these apatites, whereas loss of "H2O" between 200 degrees and 400 degrees C is irreversible and causes a contraction in the a-axis dimension. It is proposed that two general types of "H2O" are present in these apatites: (a) adsorbed H2O--characterized by reversibility, thermal instability below 200 degrees C, and lack of effect on lattice parameters; and (b) lattice H2O--characterized by irreversibility, thermal instability between 200 and 400 degrees C, and induction of expansion in the a-axis dimensions of human enamel and precipitated apatites. Lattice H2O is assumed to be due to H2O-for-OH and/or HPO4-for-PO4 substitutions in these apatites. Loss of adsorbed H2O caused sharpening of the OH absorption bands in the spectra of these apatites. Loss of lattice H2O caused the appearance of P-O-P absorption bands (due to the presence of P2O74- group) in precipitated apatites containing small amounts of CO32-.

Adult↗

Mineralogical notes of apatite in urinary calculi.

Apatite in some samples of common urinary calculi consisting mainly of apatite or of calcium oxalate and apatite was subjected to X-ray and thermal analysis and electron microscopic investigation. The X-ray analysis has determined the lattice parameters agreeing with hydroxyapatite, and the mean crystallite size normal to the basal plane in a range of 200-800 A. The thermal analysis is given by differential thermal analysis (DTA) and thermogravimetric (TG) curves. From the TG curve of a mixed apatite-calcium oxalate calculus, an approach is given to estimate the proportions of the constituent materials; the result is as follows: whewellite, 51.7%; weddellite, 16.6%; apatite, 19.1%; and organic materials, 12.6%. Analysis with the scanning electron microscope with energy dispersive X-ray spectrometer has shown that apatite is found as tiny spherules (5-10 micrometer in diameter) with fluffy surfaces resulting from projecting fine fibers or needles (about 0.5 micrometers in length). The fibers are considered to be mosaic aggregates of crystallites. The spherules fill interspaces of well-formed oxalate crystals or are included sporadically in individual crystals. Occasionally well-formed crystal-shaped oxalates are bordered with narrow bands consisting of apatite spherules (often as hemispheres) linked in a row.

Apatites↗

Poorly crystalline apatites: evolution and maturation in vitro and in vivo.

Poorly crystalline apatites (PCA) are the major mineral component of mineralized tissues in vertebrates. Their physical-chemical properties are, however, not very well known due to their relative instability and the difficulties to characterize nanocrystalline compounds. Several studies using spectroscopic techniques (Fourier transform infrared [FTIR]; 31P nuclear magnetic resonance [NMR]) have demonstrated the existence, both in precipitated and biological PCA, of labile non-apatitic environments of the mineral ions. These environments are involved in the high surface reactivity and evolution ability of PCA and they are believed to form a hydrated layer at the surface of the nanocrystals in aqueous media. The extent of the hydrated layer may vary considerably depending on the conditions of precipitation and maturation time. As PCA age, the decrease of the non-apatitic environments proportion is associated with a decrease of intracrystalline disorder and an increase of stable apatitic domains. For synthetic and biological apatites, the carbonation rate of the mineral and the uptake of essential or toxic trace elements can be related to the maturation processes. The mineral ions of the hydrated layer can be easily and reversibly substituted by other ions which can either be included in the growing stable apatite lattice during maturation or remain in the hydrated layer. In addition, the non-apatitic environments seem to be involved in the binding of soluble non-collagenic proteins. This phenomenon could be related to calcium phosphate formation; we showed that, at an albumin concentration close to that in human serum, this protein has an inhibitory effect on octacalcium phosphate crystallization on collagen in vitro.

Animals↗

Mechanism of apatite formation on wollastonite coatings in simulated body fluids.

The formation mechanism of apatite on the surface of wollastonite coating was examined. Plasma-sprayed wollastonite coatings were soaked in a lactic acid solution (pH=2.4) to result in the dissolution of calcium from the coating to form silanol (triple bond Si-OH) on the surface. Some calcium-drained samples were soaked in a trimethanol aminomethane solution (pH=10) for 24h to create a negatively charged surface with the functional group (triple bond Si-O(-)). These samples before and after treatment in a trimethanol aminomethane solution were immersed in simulated body fluids (SBF) to investigate the precipitation of apatite on the coating surface. The results indicate that the increase of calcium in the SBF solution is not the critical factor affecting the precipitation of apatite on the surface of the wollastonite coating and the apatite can only form on a negatively charged surface with the functional group (triple bond Si-O(-)). The mechanism of apatite formation on the wollastonite coating is proposed. After the wollastonite coatings are immersed into the SBF, calcium ions initially exchange with H(+) leading to the formation of silanol (triple bond Si-OH) on the surface of the layer and increase in the pH value at the coating-SBF interface. Consequently, a negatively charged surface with the functional group (triple bond Si-O(-)) forms on the surface. Due to the negatively charged surface, Ca(2+) ions in the SBF solution are attracted to the interface between the coating and solution, thereby increasing the ionic activity of the apatite at the interface to the extent that apatite precipitates on the coating surface.

Apatites↗

Mouse embryonic stem cell colonisation of carbonated apatite surfaces.

Apatites play a crucial role in the body and have been used extensively in biomedical implants. The influence on stem cell behaviour is not known and so this study will explore whether sintered carbonated apatites are favourable for propagation of stem cells. Different weight substitutions of carbonated apatite, specifically 2.5 wt% (2.5 wt%CAP) and 5 wt% (5 wt%CAP), were sintered and characterised prior to the investigation of their potential as a matrix for the support of mouse embryonic stem (ES) cells. Characterisation of the apatites included elemental analysis, X-ray diffraction, surface roughness, specific surface area, density, and solubility. The ability of carbonated apatite to support mouse ES cell colonisation and maintenance in the presence of leukaemia inhibitory factor was determined by an enumeration of live versus dead cells within a population, and immunoreactivity to Oct4, a transcription factor and stem cell marker, following growth on each matrix. It was found that while both compositions allowed for the colonisation of mouse ES cells, the cells were not maintained in an undifferentiated state, as evidenced by a reduction in the number of cells staining positive for Oct4 expression. This study shows that an increase in carbonate content within sintered apatites leads to a higher cell number, a desired aspect for stem cells to populate scaffolds intended for tissue engineering. This study presents carbonated apatites as a suitable matrix for the initial colonisation and differentiation of ES cells for tissue engineering applications.

Animals↗