Synthetic Carbonated Apatites Research Articles

Novel Synthetic Carbonate Apatite as a Bone Substitute in Implant Treatments: Case Reports.

Bone graft materials are often used in implant treatment to optimize functional and esthetic outcomes. The requirements for bone grafting materials are the ability to maintain space for bone regeneration to occur and the capability of being resorbed by osteoclasts and replaced with new bone tissue occurring in passive chemolysis and bone remodeling. Carbonate apatite (CO3Ap) granules (Cytrans Granules, GC) are a chemically synthetic bone graft material similar to autogenous bone minerals and more biocompatible than allografts and xenografts. The aim of this report is to evaluate the efficacy of CO3Ap granules in implant treatments when used alone or in combination with autogenous bone. The clinical findings and the radiographic and histologic assessments in three cases of immediate implant placement and lateral and vertical guided bone regeneration are reported. Despite the short-term follow-ups, histologic findings showed that CO3Ap granules were efficiently resorbed and replaced bone in clinical use. Furthermore, the clinical findings showed that CO3Ap granules maintained their morphology around the implant. This limited short-term case report suggests that this bone substitute is effective. However, further clinical studies and long-term reports of this new biomaterial are needed.

Unique Chemistry and Structure of Pyrolyzed Bovine Bone for Enhanced Aqueous Metals Adsorption

Bone waste is a problematic slaughterhouse waste typically disposed of in landfills. The pyrolyzed product of this waste shows strong potential in mine and industrial waste water remediation and work is needed to identify chemical and structural parameters which drive performance. Diffuse Reflectance Fourier Transform Spectroscopy (DRIFTS) was used to probe carbonate (CO32−), phosphate (PO43−) and hydroxyl (OH−) environments of mineral phases and functional group chemistry in carbonaceous phase, revealing a potentially synergistic functionality between the two in bone char. CO32− and water substitutions in the mineral lattice were found to persist after pyrolysis to 750 °C, and more soluble non-apatite calcium phosphate phases were observed using second derivative analysis of the v3 PO43− band. Nitrogen-rich functional groups were found in the carbonaceous phase which are associated with complexation of aqueous metals, and ordered aromatic clusters identified by Raman spectroscopy indicate a porous carbon skeletal structure to promote metals adsorption and complexation. These results point to unique chemical and structural features of bone char which are not easily replicated by synthetic carbonated apatite or activated carbon and which contribute to the excellent aqueous metals removal power of bone char.Graphical

Proliferation and differentiation of osteoblasts on synthetic carbonated apatite disk

Proliferation and differentiation of osteoblasts on synthetic carbonated apatite disk

Raman, FT-IR and XRD study of bovine bone mineral and carbonated apatites with different carbonate levels

A series of B-type carbonated apatites with varying carbonate levels were synthesized in order to analyze how synthetic materials and natural bone structure change with carbonate contents. FT-IR, Raman spectroscopy, and X-ray diffraction have been employed as combined tools to study the structural change of synthetic carbonated apatite and nature bone mineral. B-type carbonated apatites have been extensively investigated because they resemble most closely of mineral phase of nature bone. In this study, we found that hydroxyl ions in the apatite lattice are reduced as the carbonate contents increase. These results demonstrate that the substitution of tetrahedron phosphate ions by planar carbonate ions causes the structural deficiency of hydroxyl ions.

Effect of low-intensity pulsed ultrasound on the activity of osteoclasts: An in vitro study

ObjectiveThe objective of this in vitro study was to evaluate the effect of low-intensity pulsed ultrasound on the resorption activity of osteoclast cell cultures. DesignRAW 264.7 cells were cultured and seeded over plates that were pre-coated with a synthetic carbonate apatite, and marked with fluoresceinamine-labeled sodium chondroitin polysulfate. Plates were randomly divided into 4 groups according to the treatment assigned to each one of them: NO RANKL (no RANK-L addition and no ultrasound application), NO LIPUS (addition of RANK-L and no ultrasound application), LIPUS 10 (addition of RANK-L and 10min of ultrasound application per day), and LIPUS 20 (addition of RANK-L and 20min of ultrasound application per day). The ultrasound device produced 1.5MHz pulses with a repetition rate of 1kHz and intensity of 30mW/cm2. The experiment extended for one week and afterwards, resorption activity was evaluated according to the fluorescence intensity analysis and pit resorption measurements (number of pits and mean area). ResultsOur experiment consistently demonstrated that low-intensity pulsed ultrasound application enhanced osteoclasts resorptive activity. In addition, it was demonstrated that when daily ultrasound application lasted longer (20min) the resorption was the highest. Results obtained from both evaluation methods were reasonably coherent. ConclusionsLow-intensity pulsed ultrasound increases osteoclast resorptive activity in the absence of osteoblasts. This effect seems to be influenced by ultrasound treatment time. Future research might be directed to investigate osteoclast response to different ultrasound application protocols (frequencies and intensities) and potential cellular mechanisms.

Synthesis and Sintering-wet Carbonation of Nano-sized Carbonated Hydroxyapatite

Synthetic carbonated apatite ceramics are considered as promising alternative to auto- and allograft materials for bone substitute. In this study, Carbonated hydroxyapatite (CHA) was synthesized by nanoemulsion method. The powder produced was B-type CHA in nano-sized and had 8.25% carbonate content. The CHA samples were made into pellets and were sintered to 800°C. Upon cooling down to 150, 200, 250 and 300°C, carbonation with wet CO2 was performed on the CHA in a desiccator to re-compensate the carbonate loss due to sintering and improve densification. The aim of this study was to investigate and compare the effect of cooled down temperatures on dense CHA with two kind of wet CO2 atmospheres: direct wet CO2 and dry CO2 through water. Sintered CHA carbonated by using dry CO2 through water had overall higher amount of carbonate content as compared to carbonation from wet CO2 directly from tank. D200, sample undergone carbonation by carbonated by dry CO2 through water at 200°C had the highest carbonate content (3.35%).

The Newly Bone Formation with Carbonate Apatite-Chitosan Bone Substitute in the Rat Tibia

Large bone defect still represent a major problem in orthopedics. A tissue engineering approach has been proposed where osteogenic cells, bioceramic scaffolds and growth factors can play in a role to the bone repair. Bone consist a mineral phase such as carbonate apatite and an organic phase such as collagen. Synthetic carbonate apatite ceramics are considered as promising alloplastic materials for bone substitute. Chitin is the organic matrix of the hard parts of exoskeleton of insect, crustacean and present in a small amounts in mushrooms. It is an insoluble, similar to cellulose and composed of N-acetylglucosamine unit. Partial deacetylation from chitin result in the formation of chitosan. Chitin’s properties as a flexible and strong material make it favourable as surgical thread. It has novel properties such as biocompatibility, biodegradability, anti bacterial, wound healing activity, tissue regeneration and hemostatic activitities. The composit from carbonate apatite and chitosan may have a great impact on human health care system as bioresorbable bone substitute. The aim of the study was to evaluate the newly bone formation on the bone healing of defect tibia treated with carbonate apatite-chitosan bone substitute. Eighteen Sprague Dawley rats, male, 3 months, weighing 250-300g used in this study. Bilateral defect were created in each tibia rat. The defects were filled with carbonate-apatite chitosan bone substitute. The rats were sacrificed after respectively 1, 2 and 3 weeks. The result of this study showed that carbonate apatite-chitosan significantly increased a number of osteoblast (p<0.05). Carbonate apatite-chitosan group showed that matrix deposition faster than the other groups and have a good interface with the old bone. These data indicate that carbonate apatite-chitosan are potential candidate for bone substitute

OPTIMIZING OF NEW APPROACH SINTERING METHOD ON CARBONATED HYDROXYAPATITE BIOCERAMIC

Synthetic carbonated apatite ceramics are considered as alternative to autograft and allograft materials for bone substitute. This study was to investigate and compare the effect of carbonation introduced during cooling down of sintering of dense CHA with two kind of wet CO2 atmosphere (direct wet CO2 and dry CO2 flow through water). Pelletized samples were sintered to 800°C and soaked for 120 minutes. Upon cooling down to 150, 200, 250 and 300°C, carbonation with wet CO2 in desiccators were performed. The sintered CHA after carbonation at the various cooled temperatures was found to retain as B-type CHA. Carbonation of sintered CHA by dry CO2 through water showed an overall better results as compared to carbonation by direct wet CO2. Sample D200 with carbonation temperature of 200oC has the highest carbonate content (3.35%) and relative density (94%) while D250 with carbonation temperature of 250oC has the highest DTS value (18MPa). In vitro testing using SBF solution showed that apatite layer formed on the sintered CHA surface after one week of immersion, thus indicating the bone-forming ability. This indicated that the CHA synthesized in this study has sufficient biocompatibility to be applied as bone substitutes.

Statistical Studies of the Physicochemical Analytic Results of a Series of Synthetic Calcium Hydroxyapatite Containing Carbonate and Sodium

The objective of this study is to present a simple method of statistical calculation that allowed us to determine the relationship between the different data obtained from the characterization of the synthetic carbonated apatites containing sodium, in order to find the fundamental substitution mechanism(s) for incorporation of Na+ and CO32- and to establish the general formula. For that, a series of hydroxyapatites containing carbonate and sodium (Na-CO3HAps) has been obtained by the precipitation method. All the compounds were characterized by infrared spectra (IR), powder X-ray diffraction (PXRD) and elemental analysis. The statistical treatment of the experiment result allows us to determine the relationship between one variable and the change in the other and to found the fundamental substitution mechanism(s) for incorporation of Na+ and CO32- . Analysis of variance (ANOVA) allows us to test the models proposed.

Effect of carbonate incorporation on the hydroxyl content of hydroxylapatite

AbstractMechanisms for the incorporation of carbonate into minerals of the apatite group have been explored in both the geological and medical literature. An important problem with respect to biological apatite, which requires further clarification, is the hydroxyl content of the carbonated apatite of bone. Recent studies reveal bone apatite to contain only ∼20 mol.% of the hydroxyl content of stoichiometric hydroxylapatite, with negligible chloride or fluoride. We investigated the hypothesis that the development of vacancies in the hydroxyl channel sites is a charge-balancing mechanism for the substitution of carbonate ions into hydroxylapatite. Raman spectroscopic analyses of synthetic carbonated apatites (containing 1 to >15 wt.% carbonate) show that their hydroxyl ion concentration correlates inversely with carbonate concentration. The specific relationship between carbonate and hydroxyl concentration in these samples closely follows the theoretical relationship defined by type-B substitution of carbonate for phosphate in the apatite structure. However, the 6–8 wt.% carbonate concentration in bone apatite falls far short of accounting for all of the hydroxyl depletion that occurs in bone apatite. Some of the additional hydroxyl depletion in bone apatite might result from substitution of Na+ for Ca2+, but further mechanism(s), perhaps (HPO4)2– substitution, must also play a significant role.

A Comparative Study of Carbonate Determination in Human Teeth Using Raman Spectroscopy

Carbonate determination in dental apatites such as dentine and enamel is important for studying the dynamics of dental caries and developmental defects of these tissues. Traditionally, these determinations have been performed by acidic digestion with the subsequent measurement of released carbon dioxide gas. As an alternative, Raman spectroscopy has been used for the determination of carbonate in synthetic carbonated apatites with at least four analytical methods published thus far. However, these methods have not been applied to biological apatites. The aim of this comparative study was to test the suitability of these four methods for the determination of B-type carbonate in human enamel and dentine. A method for determining the A-type carbonate content of enamel using the Raman technique is also presented. Raman spectra were obtained from 10 human enamel and dentine samples and analysed with each of the four methods using either a single or multiple ν₁(PO₄^3–) band spectral fitting model. Each of the methods resulted in a different determination for the carbonate content when using the same measurement data. The method that used the full-width-at-half-maximum of the ν₁(PO₄^3–) band to determine the B-type carbonate concentration was found to be in best agreement with (i) the results (using the acid digestion method) of teeth collected from the same sample population and (ii) previously reported values for both enamel and dentine. The use of a multiple-band spectral fitting model produced the highest determination precision (particularly in the case of dentine).

A new possible atomic arrangement for the carbon atom in the B-sites of AB-type carbonate apatite

Received: March 17, 2011; Revised: July 21, 2011The crystal structure of a synthetic AB-type carbonate apatite sample was analyzed by Rietveld refinement including a model with carbon atom not fixed in the B-site of the apatite structure. Only one constraint was applied to this model: the fractional occupancies of the atoms in the CO

Comparison of a synthetic bone substitute composed of carbonated apatite with an anorganic bovine xenograft in particulate forms in a canine maxillary augmentation model

To evaluate the effect of a porous geometry in particulate bone on new bone formation by comparison of anorganic bovine carbonate apatite (ABCA) with synthetic carbonated apatite (SCA), which have similar properties but different micro-structures. Porous structures and anorganic components of ABCA and SCA were evaluated using scanning electron microscope and Fourier transform infrared. They were implanted in maxillary augmentation models with the mouth split design in a total of 15 Beagle dogs. The animals were sacrificed 4, 8 and 16 weeks after surgery, and the histomorphometrical results were statistically analyzed for the material's geometrical relationship and new bone formation in relation to the available space and contact surface for osteoconduction. Both materials showed a typical infrared pattern of CO(3)(2-) -substituted hydroxyapatite (HA). Porous structures and a bridging effect of osteoconductive bone material were relatively better observed in SCA. The ratio of the material area to the total area was higher (P<0.01) for ABCA (28.03±6.09) than for SCA (20.26±4.23). The ratio of the number of particles possessing a pore structure to the total number and the interparticular space was greater (P<0.001 and 0.01) for SCA (18.12±9.44 and 79.74±4.23) compared with ABCA (1.45±1.74 and 71.63±5.85). The new bone areas and the bone-material contact lengths were greater in SCA than in ABCA (P<0.05). The present study showed that porous structures may have an influence on new bone formation in osteoconductive bone substitutes.

Porcine model for hybrid material of carbonated apatite and osteoprogenitor cells

Culture and characterisation of porcine osteoprogenitors were established in order to study the differentiation of osteoblast in a hybrid material of carbonated apatite (CAp) (patent under process no. PI 20042321) and autologous cells that can be used later as an advanced bone graft material. Cells were obtained from bone marrow and cultured onto CAp of wet synthesis and onto Thermanox, with or without differentiation factors in time course experiments, at the end of which biochemical and molecular markers of osteogenesis were obtained. Results indicated that this model is useful to study the functionality of a hybrid material of synthetic CAp and autologous osteoprogenitor cells derived from porcine bone marrow.

Nanosized particles in bone and dissolution insensitivity of bone mineral

Most of the mineral crystals in bone are platelets of carbonated apatite with thicknesses of a few nanometers embedded in a collagen matrix. We report that spherical to cylindrical shaped nanosized particles are also an integral part of bone structure observed by high resolution scanning electron microscopy. High resolution back scattered electron imaging reveals that the spherical particles have a contrast similar to the crystal platelets, suggesting that they are thus likely to have similar mineral properties. By means of constant composition (CC) dissolution of bone, similar sized nanoparticles are shown to be insensitive to demineralization and are thought to be dynamically stabilized due to the absence of active pits/defects on the crystallite surfaces. Similar reproducible self-inhibited dissolution was observed with these nanoparticles during CC dissolution of synthetic carbonated apatite. This result rules out the possible influence of complicating biological factors such as the possible presence of organic matrix components and other impurities. This phenomenon can be explained by a unique dissolution model involving size considerations at the nanoscale. The unexpected presence of nanoparticles in mature bone may also be due to the stabilization of some nanosized particles during the formation process in a fluctuating biological milieux.

Carbonate release from carbonated hydroxyapatite in the wide temperature rage

Synthetic carbonated apatite ceramics are considered as promising alternative to auto- and allograft materials for bone substitute. The aim of this study was to investigate the thermal stability of an AB-type carbonated apatite in the wide temperature range. The data on the thermal stability have to allow the conditions of the sintering of the ceramics to be controlled. Initial carbonated apatite powders were prepared by interaction between calcium oxide and ammonium hydrogen phosphate with addition of ammonium carbonate. Decomposition process was monitored by infra red spectroscopy, weight loss and X-ray diffraction of solid, and by infra red analysis of condensed gas phase resulted from the thermal decomposition of the sample in equilibrium conditions. Features of carbon monoxide and carbon dioxide release were revealed. The synthesized AB-type carbonated apatite is started to decompose at about 400 degrees Celsius releasing mainly carbon dioxide, but retained some carbonate groups and apatite structure at the temperature 1100 degrees Celsius useful to prepare porous carbonate-apatite ceramics intended for bone tissue engineering scaffolds.

Three Structural Roles for Water in Bone Observed by Solid-State NMR

Hydrogen-bearing species in the bone mineral environment were investigated using solid-state NMR spectroscopy of powdered bone, deproteinated bone, and B-type carbonated apatite. Using magic-angle spinning and cross-polarization techniques three types of structurally-bound water were observed in these materials. Two of these water types occupy vacancies within the apatitic mineral crystal in synthetic carbonated apatite and deproteinated bone and serve to stabilize these defect-containing crystals. The third water was observed at the mineral surface in unmodified bone but not in deproteinated bone, suggesting a role for this water in mediating mineral-organic matrix interactions. Direct evidence of monohydrogen phosphate in a 1H NMR spectrum of unmodified bone is presented for the first time. We obtained clear evidence for the presence of hydroxide ion in deproteinated bone by 1H MAS NMR. A 1H-31P heteronuclear correlation experiment provided unambiguous evidence for hydroxide ion in unmodified bone as well. Hydroxide ion in both unmodified and deproteinated bone mineral was found to participate in hydrogen bonding with neighboring water molecules and ions. In unmodified bone mineral hydroxide ion was found, through a 1H-31P heteronuclear correlation experiment, to be confined to a small portion of the mineral crystal, probably the internal portion.

Highly Ordered Interstitial Water Observed in Bone by Nuclear Magnetic Resonance

NMR was used to study the nanostructure of bone tissue. Distance measurements show that the first water layer at the surface of the mineral in cortical bone is structured. This water may serve to couple the mineral to the organic matrix and may play a role in deformation. The unique mechanical characteristics of bone tissue have not yet been satisfactorily connected to the exact molecular architecture of this complex composite material. Recently developed solid-state nuclear magnetic resonance (NMR) techniques are applied here to the mineral component to provide new structural distance constraints at the subnanometer scale. NMR dipolar couplings between structural protons (OH(-) and H(2)O) and phosphorus (PO(4)) or carbon (CO(3)) were measured using the 2D Lee-Goldburg Cross-Polarization under Magic-Angle Spinning (2D LG-CPMAS) pulse sequence, which simultaneously suppresses the much stronger proton-proton dipolar interactions. The NMR dipolar couplings measured provide accurate distances between atoms, e.g., OH and PO(4) in apatites. Excised and powdered femoral cortical bone was used for these experiments. Synthetic carbonate ( approximately 2-4 wt%)-substituted hydroxyapatite was also studied for structural comparison. In synthetic apatite, the hydroxide ions are strongly hydrogen bonded to adjacent carbonate or phosphate ions, with hydrogen bond (O-H) distances of approximately 1.96 A observed. The bone tissue sample, in contrast, shows little evidence of ordered hydroxide. Instead, a very ordered (structural) layer of water molecules is identified, which hydrates the small bioapatite crystallites through very close arrangements. Water protons are approximately 2.3-2.55 A from surface phosphorus atoms. In synthetic carbonated apatite, strong hydrogen bonds were observed between the hydroxide ions and structural phosphate and carbonate units in the apatite crystal lattice. These hydrogen bonding interactions may contribute to the long-range stability of this mineral structure. The biological apatite in cortical bone tissue shows evidence of hydrogen bonding with an ordered surface water layer at the faces of the mineral particles. This structural water layer has been inferred, but direct spectroscopic evidence of this interstitial water is given here. An ordered structural water layer sandwiched between the mineral and the organic collagen fibers may affect the biomechanical properties of this complex composite material.

Ultrastructural analysis of structural framework in dental plaque developing on synthetic carbonate apatite applied to human tooth surfaces.

This study focused on determining the structural framework by oral microbiota in supragingival plaque on a carbonate apatite film applied to human tooth surfaces. The sequential phases of plaque formation over a 3-wk period were found to be equivalent to those previously reported for natural tooth surfaces. Scanning electron microscopy of specimens prepared by vertical sectioning demonstrated the organization of two types of framework between certain genera of initial and secondary colonizers in the pre- and post-organization phases, respectively. The initial colonizers in the pre-organization phase were of a coccoid type, while colonizers in the post-organization phase were of a bacillary type. Secondary colonizers, filamentous cells, were common to both frameworks. Transmission electron microscopy using freeze-substitution and immunohistochemistry demonstrated two types of coaggregation, fibril- and saliva-mediated modes, among the plaque microbiota. Coaggregation between microbiota, which organized the framework, showed a tendency to occur in the fibril-mediated mode, and the filamentous secondary colonizers were characterized by inducing multigeneric coaggregation. The present findings indicate that a structural framework and specific cells to form this framework are essential for plaque formation.

Infrared microscopic imaging of bone: spatial distribution of CO3(2-).

This article describes a novel technology for quantitative determination of the spatial distribution of CO3(2-) substitution in bone mineral using infrared (IR) imaging at approximately 6 microm spatial resolution. This novel technology consists of an IR array detector of 64 x 64 elements mapped to a 400 microm x 400 microm spot at the focal plane of an IR microscope. During each scan, a complete IR spectrum is acquired from each element in the array. The variation of any IR parameter across the array may be mapped. In the current study, a linear relationship was observed between the band area or the peak height ratio of the CO3(2-) v3 contour at 1415 cm(-1) to the PO4(3-) v1,v3 contour in a series of synthetic carbonated apatites. The correlation coefficient between the spectroscopically and analytically determined ratios (R2 = 0.989) attests to the practical utility of this IR area ratio for determination of bone CO3(2-) levels. The relationship forms the basis for the determination of CO3(2-) in tissue sections using IR imaging. In four images of trabecular bone the average CO3(2-) levels were 5.95 wt% (2298 data points), 6.67% (2040 data points), 6.66% (1176 data points), and 6.73% (2256 data points) with an overall average of 6.38+/-0.14% (7770 data points). The highest levels of CO3(2-) were found at the edge of the trabeculae and immediately adjacent to the Haversian canal. Examination of parameters derived from the phosphate v1,v3 contour of the synthetic apatites revealed that the crystallinity/perfection of the hydroxyapatite (HA) crystals was diminished as CO3(2-) levels increased. The methodology described will permit evaluation of the spatial distribution of CO3(2-) levels in diseased and normal mineralized tissues.