Calcium-deficient Apatite Research Articles

Fabrication of novel bioresorbable .BETA.-tricalcium phosphate cement on the basis of chelate-setting mechanism of inositol phosphate and its evaluation

We have developed novel bioresorbable β-tricalcium phosphate (β-TCP) cements on the basis of chelate-setting mechanism of inositol phosphate (IP6). The starting cement powders (IP6/β-TCP powders) were prepared by surface-modifying β-TCP particles with IP6. The cement specimen was fabricated by mixing the IP6/β-TCP powder in pure water at desired powder/liquid ratios, and examined the effects of powder properties of the IP6/β-TCP powder on the mechanical strength (compressive strength) of the cement specimens. We focused on the crystalline phase, particle size, specific surface area (SSA), and crystallite size among powder properties. The crystalline phase of resulting cement specimen was β-TCP single phase or mixture of β-TCP and calcium-deficient apatite (CDAp). The β-TCP cement with compressive strength of 13 MPa was fabricated from the finely-ground β-TCP powders prepared by ball-milling commercially-available β-TCP powder for 4 h using zirconia beads with 10 mm in diameter. Meanwhile, the β-TCP/CDAp biphasic cement had maximum compressive strength of about 23 MPa among the examined cement specimens, it was fabricated from the ball-milled commercially-available β-TCP powder for 3 h using zirconia beads with 10 mm in diameter, and then for 3 h using zirconia beads with 2 mm in diameter. In order to make the determining factors of the compressive strength clear, we examined the relationship between powder properties (particle size, SSA and crystallite size) and compressive strength. The strength of the IP6/β-TCP cement was not dependent on the particle size of the IP6/β-TCP powder; meanwhile, the strength was enhanced with increasing SSA and decreasing crystallite size. Thus, the IP6/β-TCP powder with higher SSA and smaller crystallite size may be useful in the fabrication of chelate-setting β-TCP cement with enhanced mechanical properties.

Intrafibrillar collagen mineralization produced by biomimetic hierarchical nanoapatite assembly.

Biomineralization templated by non-collagenous proteins is an example how nature uses nanotechnology to strengthen the load-bearing skeletons of vertebrates.[1–3] Mineralized collagen fibrils are responsible for the second level of hierarchy of bone structure[4] and account for its strength and toughness.[5] Calcium-deficient apatites are deposited in nanocrystalline form at the intrafibrillar and extrafibrillar spaces associated with the collagen fibrillar assembly.[6] Regulation of calcium phosphate phases during biomineralization is believed to exhibit two major characteristics.[7–9] The first is the sequestration of the calcium and phosphate ions into nanoscopic entities known as amorphous calcium phosphate (ACP). The second is the templating of mineral nucleation and growth through noncollagenous extracellular matrix proteins. These protein molecules contain polyanionic domains that are rich in polycarboxylic acid and phosphate function groups that bind to the collagen substrates at specific sites such as the gap zones of the collagen molecular assembly.[1,10] The immobilized proteins contain anionic charge sites wherein calcium binding and apatite nucleation occur at the aforementioned intrafibrillar locations. As a highly ordered manner of intrafibrillar mineralization at the nanoscale is responsible for the biomechanical properties of mineralized collagen,[11] this intricate biological process has inspired scientists to mimic its mechanism for creating mineralized type I collagen scaffolds to repair existing bone defects and/or generate new bone. Here, we demonstrate that the use of two phosphate-based templating analogs of matrix proteins in the presence of a polycarboxylic acid sequestration analog of ACPs results in a highly ordered intrafibrillar nanoapatite assembly that recapitulates the gap and overlap arrangement of collagen molecules within a collagen fibril. Adoption of a dual analog biomimetic strategy provides a means to produce advanced mineralized collagen scaffolds for tissue engineering applications.

공침법을 이용한 마그네슘이 첨가된 biphasic calcium phosphate의 적합성 평가

Mg-BCP(Mg Substituted BCP)를 제조하기 위하여 <TEX>$Ca(NO_3)_2{\cdot}4H_2O$</TEX>(Katayama chemical, Japan)과 <TEX>$(NH_4)_2{\cdot}HPO_4$</TEX> (Junsei chemical, Japan), <TEX>$Mg(NO_3)_2{\cdot}6H_2O$</TEX>(Junsei chemical, Japan)를 출발물질로 공침법(co-precipitation process)을 이용하여 합성하였다. 제조된 분말의 마그네슘의 첨가에 따른 분말의 분광학적 특성은 FT-IR(MAGNA-IR 560, Nicolet)을 통하여 분석하였으며, SEM(S-4200, Hitachi)을 통하여 미세구조를 분석하였다. XRD 회절피크의 면적적분강도를 Integral Analysis (Rigaku, Japan)를 이용하여 HAp와 <TEX>${\beta}$</TEX>-TCP의 비율을 확인하였다. 1.0 wt% 마그네슘이 첨가된 BCP 샘플의 경우 Hank's solution에서의 2주 침적 후 표면에 침상의 HAp로 추정되는 결정이 성장하였다. 이러한 미세구조의 변화는 생활성을 가지는 마그네슘의 첨가가 BCP 조직 내에서 표면의 활성을 증가시켜 결정의 성장을 촉진시킨 것으로 판단된다. Magnesium-substituted BCP (biphasic calcium phosphate) powders were prepared by incorporating small amounts of magnesium into the structure of different hydroxyapatite (HAp)/<TEX>${\beta}$</TEX>-tricalcium phosphate (<TEX>${\beta}$</TEX>-TCP) ratios through coprecipitation method. A series of magnesium substitutions ranging from 0, 0.5, and 1.0 wt%, which are comparable to the measured magnesium contents, were performed. The obtained powders were characterized by the following analytical techniques: X-ray diffraction analysis (XRD), Thermo Gravimetric Analyzer (TGA) and Fourier transform infrared spectroscopy (FT-IR). The results have shown that substitution of magnesium in the calcium-deficient apatites resulted in the formation of biphasic mixtures of different HAP/<TEX>${\beta}$</TEX>-TCP ratios after heating above <TEX>$1000^{\circ}C$</TEX>. The 1.0 wt% magnesiumsubstituted-BCP were soaked in Hank's solutions after 2 weeks to observe the morphology of the biocement, especially needle-like hydroxyapatite crystals and to estimate the length and diameter of nanoneedle crystals.

In Vitro Characterization of Calcium Phosphate Biomaterial Loaded with Linezolid for Osseous Bone Defect Implantation

Osteomyelitis is a severe bone infection frequently caused by Staphylococcus aureus, which shows significant resistance to methicillin. One therapeutic treatment would be to insert a bone substitute loaded to an antibiotic, which would enable the bone to be filled while the illness is being treated. Linezolid is an oxazolidinone antibiotic with a large spectrum of action. It is effective against most Gram-positive bacteria and displays a specific mode of action. The aim of this work was to study the association of linezolid with a calcium phosphate-deficient apatite matrix. Granules containing 10% and 50% linezolid were prepared by wet granulation and characterized. Porosity analyses performed by mercury porosimetry and scanning electron microscopy revealed that grain porosity with 50% linezolid was higher than that of the grains containing 10% linezolid. NMR analyses showed no change in structure of linezolid when linked to calcium-deficient apatite. These results were confirmed by studying the antibacterial activity of linezolid, which remained proportional to the quantity of loaded linezolid, proving that the antibiotic released was active. The in vitro release time varied from 9 days for granules containing 10% linezolid to 26 days for granules containing 50% linezolid.

Investigation of alendronate-doped apatitic cements as a potential technology for the prevention of osteoporotic hip fractures: Critical influence of the drug introduction mode on the in vitro cement properties

Combination of a bisphosphonate (BP) anti-osteoporotic drug, alendronate, with an apatitic calcium phosphate cement does not significantly affect the main properties of the biomaterial, in terms of injectability and setting time, provided that the BP is introduced chemisorbed onto calcium-deficient apatite, one of the components of the cement. In contrast to other modes of introducing the BP into the cement formulation, this mode allows to minimize alendronate release in the cement paste, thus limiting the setting retardant effect of the BP. An original approach based on high frequency impedance measurements is found to be a convenient method for in situ monitoring of the cement setting reaction. The release profile of the drug from a cement block under continuous flow conditions can be well described using a coupled chemistry/transport model, under simulated in vivo conditions. The results show that the released alendronate concentration is expected to be much lower than the cytotoxic concentration.

Analgesic properties of calcium phosphate apatite loaded with bupivacaine on postoperative pain

Synthetic calcium-deficient apatites (CDA) are structurally similar to biological apatites and are well known as chemical precursors of biphasic calcium phosphates (BCP). BCP are mixtures of hydroxyapatite and beta-tricalcium phosphate and are widely used as bone substitutes in human surgery. Bupivacaine, a local anesthetic, has been loaded onto CDA using isostatic compaction. The purpose of this study was to evaluate the in vivo performance of such a local release on pain after having previously defined the in vitro release profile of bupivacaine. CDA was loaded with 1%, 4%, and 16% of bupivacaine using an isostatic compaction process. In vitro release profile assays performed indicated the complete release of bupivacaine after 24 h. Wistar male rats received 50 mg implants of CDA associated, respectively, with 0, 1%, 4%, and 16% of bupivacaine into the distal femur. Analgesia was measured using the electronic version of the Von Frey monofilament test, assessing the inflammatory response and a neurological score. During the first postoperative days, a dose-dependent analgesic effect was observed with the bupivacaine adsorbed on the resorbable implant. This combined device system thus appears to release local anesthetic in a manner that prevents or limits postoperative pain following bone surgery. This innovative approach could be integrated into a global pain management program, for example, in the context of bone harvesting where bone reconstruction is required.

Effect of Fluoride-substituted Apatite on In Vivo Bone Formation

Biological apatites are characterized by the presence of minor constituents such as magnesium (Mg), chloride (Cl), or fluoride (F) ions. These ions affect cell proliferation and osteoblastic differentiation during bone tissue formation. F-substituted apatites are being explored as potential bonegraft materials. The aim of the present study is to investigate the mechanism of bone formation induced by fluoride-substituted apatite (FAp) by analyzing the effect of FAp on the process of in vivo bone formation. FAps containing different F concentrations (l-FAp: 0.48 wt%, m-FAp: 0.91 wt%, h-FAp: 2.23 wt%) and calcium-deficient apatite (CDA), as positive control, were implanted in rat tibia and bone formation was evaluated by histological examination, immuhistochemistry, in situ hybridization and tartrate-resistant acid phosphatase examinations. The results showed that l-FAp, m-FAp, h-FAp, and CDA biomaterials allowed migration of macrophages, attachment, proliferation, and phenotypic expression of bone cells leading to new bone formation in direct apposition to the particles. However, the l-FAp preparation allowed faster bone conduction compared to the other experimental materials. These results suggest that FAp with low F concentration may be an efficient bonegraft material for dental and medical application.

Development of bisphosphonates controlled delivery systems for bone implantation: influence of the formulation and process used on in vitro release

The present study investigates the development of controlled drug delivery devices by association of bisphosphonates (BPs) with calcium-deficient apatite (CDA) to obtain a prolonged drug delivery. In a first part, we studied the microencapsulation of methylene bisphosphonic acid, our model of BPs, in biodegradable PLGA by the double emulsion (w/o/w) solvent evaporation/extraction process. Secondly, we associated BPs, either in a free form or microencapsulated, with calcium phosphate biomaterials. The association of free BPs with CDA was performed by isostatic compression at 80 MPa and we tested the interest of adding a binder, HPMC, in the formulation to reinforce the association. In parallel, microparticles were associated with calcium-deficient apatite, either by simple mixture or by isostatic compression. To compare the different formulations, in vitro dissolution studies were performed. All the formulations tested appear to be efficient to produce BPs loaded biomaterials able to deliver the drug slowly and at a constant rate. The slowest release rate (2.7% in 14 days) was obtained with the blend of microencapsulated BPs with CDA.

Synthesis and structural characterization of strontium- and magnesium-co-substituted β-tricalcium phosphate

The synthesis of five different Sr 2+- and Mg 2+-co-substituted β-tricalcium phosphate (β-TCP) has been obtained by heating the calcium-deficient apatites above 800 °C. With the investigated concentrations of Sr 2+ and Mg 2+ from the present study, no additional phases other than β-TCP have been detected. The synthesized powders have been characterized by X-ray diffraction, Fourier transform infrared spectrometry, elemental analysis and Rietveld refinement studies. The co-substitution of Sr 2+ and Mg 2+ in the β-TCP has resulted in the formation of crystalline β-TCP at hexagonal setting (space group R3 c). The reduction of lattice a- and c-axis parameters with the combined substitution of Sr 2+ and Mg 2+ in the β-TCP has been found evident from the present results. Sr 2+ has been found occupying the Ca(1,2,3,4) sites and Mg 2+ was found at the sixfold coordinated Ca(5) site of β-TCP structure.

Local management for post operative by associating calcium deficient apatite with a local anesthetic

Local management for post operative by associating calcium deficient apatite with a local anesthetic

Effects of pH and Ca/P molar ratio on the quantity and crystalline structure of calcium phosphates obtained from aqueous solutions

Milk and dairy products contain large amounts of calcium phosphate salts that can precipitate. The chemical composition and the crystalline structure of the calcium phosphate precipitates that are formed in dairy industry depend on the physico-chemical conditions, particularly, pH and mineral composition. The objective of this study was to determine, using mineral solutions, the effects of pH and of the concentrations of calcium and phosphate on the quantity and crystalline structure of calcium phosphate precipitates. Experiments were carried out at 20 °C with 20.00 mmol·L−1 phosphate and three Ca/P molar ratios (1.00, 1.50 and 2.00). The initial pH (5.50, 6.70, 7.50, 8.50 and 9.50) were drifting or kept constant during a reaction time of 3 h. After filtration of the suspensions, the mineral compositions of filtrates were quantified. The lyophilized precipitates were characterized using X-ray diffraction, infrared spectroscopy and scanning electron microscopy techniques. At drifting pH (final pH values were between 4.6 and 6.0), the mineral analyses showed that the Ca/P ratio did not influence the amounts of precipitated calcium and phosphate. The analyses of precipitates revealed the formation of brushite as the main crystalline phase. At constant pH, the mineral analyses showed that the Ca/P ratio strongly influenced the precipitation efficiency of calcium phosphate. The analyses of precipitates revealed the formation of poorly crystallized calcium-deficient apatites. A decrease of crystallinity with an increase in initial pH was observed. In conclusion, pH can be a key factor to control the quantity and crystalline structure of calcium phosphates obtained by precipitation. This factor should be considered for the recovery of calcium phosphates from dairy co-products. pH is also important in the fouling phenomena of membranes and heat exchangers caused by calcium phosphate precipitation.

Calcium phosphate-based particles influence osteogenic maturation of human mesenchymal stem cells

Biphasic calcium phosphates (BCPs) consist of a mixture of hydroxyapatite and β-tricalcium phosphate and are recommended as alternatives or additives to autogenous bone for orthopaedic and dental applications. There is clinical evidence showing particle release from bioceramics, which might impair the ability of human mesenchymal stem cells (hMSC) from bone marrow to proliferate or mature into a functional osteoblast phenotype. This study analyses the influence of BCP particles and their precursors, calcium-deficient apatite (CDA) particles, on in vitro hMSC behaviour. Both types of particles were efficiently internalized by hMSC. Cell viability, morphology and actin cytoskeleton reorganization were unaffected by exposure of hMSC to BCP or CDA particles. Direct exposure to BCP particles impaired hMSC osteogenic differentiation and bone matrix mineralization to a lesser extent than CDA, as assayed by evaluation of alkaline phosphatase activity, osteopontin secretion and mineralized nodule formation. The ability of bioceramic particles to affect osteogenic maturation through modification of soluble factors in media was assayed in an in vitro system that avoids direct cell–particle contact. Indirect exposure to CDA particles severely impaired hMSC osteogenic maturation owing to the uptake of Ca 2+ from the culture media. Lower textural properties of BCP and the lack of calcium deficiency in its composition prevented Ca 2+ uptake, allowing the development of a functional osteoblast phenotype.

Human osteoblasts adhesion and proliferation on magnesium-substituted tricalcium phosphate dense tablets

Tricalcium phosphate (TCP) is recognized as a promising bone replacement material due to its high bioactivity and resorbable properties. To mimic biological apatites, incorporation of magnesium (Mg) in TCP was proposed. Mg-substituted TCP (beta-TCMP) and beta-TCP dense tablets were obtained by pressing and sintering at 1,000 degrees C Mg-substituted calcium deficient apatite (Mg-CDA) and commercial TCP, respectively. The materials were characterized using X-ray diffraction, infrared spectroscopy and electron microscopy. Human osteoblast cells (SaOs2) were seeded onto the sintered tablets for 4 h, 24 h and 7 days. Results showed that Mg-CDA was completely transformed into beta-TCMP. Moreover, beta-TCMP stimulated adhesion and proliferation of human osteoblast cells. Consequently, the magnesium incorporation on calcium deficient apatites followed by sintering at 1,000 degrees C seems to be a useful path to obtain biocompatible and non cytotoxic dense tablets with TCP structure with potential application on bone engineering.

Single-Component Self-Setting Calcium Phosphate Cement Concept

Single-component, self-setting and injectable calcium phosphate cement (CPC) based on amorphization process of dicalcium phosphate dehydrate (DCPD) is proposed. After preparation of DCPD by wet chemistry, the material was dry milled in an Attritor high energy process (at 400 RPM) during 20 minutes. Experiments were also conducted using a regular ball milling process at 15 and 30h residence time. Amorphization of DCPD confirmed using FTIR, XRD and 31 P solid-states NMR (cross-polarization and decoupling). Upon hydration of amorphized DCPD powder with saline (0.55 ml/g), putty-like consistency produced. The paste hardened in 30 minutes at 37°C and reached a compressive strength of about 20 MPa. The final product was a low crystalline calcium deficient apatite, similar to the composition and structure of bone mineral.

Rietveld Refinement of Sintered Magnesium Substituted Calcium Apatite

The incorporation of magnesium in the synthetic apatite has been associated with biomineralization process and osteoporosis therapy in human and animals. Magnesium easily replaces calcium in the apatite lattice and influences or controls the hydroxyapatite crystallization processes. In this work, Mg-substituted calcium deficient apatite, with Mg/Ca ratio = 0.1, 0.15 and 0.2 were synthesized by precipitation method. Then, sintered at 1000 oC and compared with a commercial product labeled as tricalcium phosphate sintered at the 1000 oC. The sintered products showed tricalcium phosphate (β-TCP) structure. The Mg2+ substitution in the Ca(4) and Ca(5) sites of β-TCP and the lattice parameter changes were estimated using the Rietveld method. Using this method, the formulas Ca2.73(Mg0.27)(PO4)2, Ca2.71(Mg0.29)(PO4)2 and Ca2.70(Mg0.23Mg0.07)(PO4)2 were calculated for the samples with Mg/Ca ratio = 0.1, 0.15 and 0.2 respectively.

Characterization of Nano-Biphasic Calcium Phosphates Synthesized under Microwave Curing

Two biphasic BCP ceramic samples were synthesized by chemical precipitation and microwave curing of calcium deficient hydroxyapatite CDHA under the same pH value and temperature but varied in their initial Ca/P molar ratio. Precipitates were characterization after thermogravimetric analysis, fourier transform infrared spectroscopy, X-ray diffraction, atomic absorption spectroscopy and TEM. Hydroxyapatite (HA) contents were measured for the two biphasic calcium phosphate (BCP) ceramics by sintering the calcium-deficient apatites (CDHA). The results reveal two condensation mechanisms of HPO42- affecting the Ca/P molar ratio after calcination. The X-ray diffraction patterns of BCP powders show the in situ formation of -TCP in the BCP powder. The amount of -TCP phase increases as the initial Ca/P molar ratio decreases due to more calcium deficiency in CDHA structure. The influence of HPO42- incorporation on increasing -TCP phase content after calcination is evaluated. TEM micrographs proved the effect of microwave curing during the preparation process on reducing of particle size to nanoscale range and the destruction of CDHA to finer HA and -TCP particles upon calcination.

Calcium orthophosphates: crystallization and dissolution.

Calcium orthophosphates: crystallization and dissolution.

Preparation and characteristics of nanosized carbonated apatite by urea addition with coprecipitation method

Nanosized carbonated apatites (CAp) were prepared by coprecipitation method with urea addition at pH 8 and pH 12 with 12 hours aging, which resulted in needle-like and rod-like structures, respectively. From examining the XRD patterns, it was determined that the as-prepared powders contained no crystalline phase other than CAp. However, the powder prepared at pH 8 partially transforms to β-tricalcium phosphate (β-TCP) after heat treatment at 900 °C. Under heat treatment, powder prepared at pH 12 is more stable than powder prepared at pH 8. In addition, the as-prepared powders at pH 8 with 12 hours aging yield needle-like calcium deficient apatite (CDA) with substitution of CO 3 2− for PO 4 3− in low content. Their particle size is about 20 nm in width and 200–300 nm in length. The as-prepared powders at pH 12 for 12 hours aging result in rod-like nanostructures about 50 nm in diameter and 300 nm in length with 8.2 wt.% CO 3 2−, which is similar to human bone composition. Various characteristic analyses of CAp have been employed including X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), energy dispersive spectrometer (EDS), and transmission electron microscopy (TEM).

Controlled release of bisphosphonate from a calcium phosphate biomaterial inhibits osteoclastic resorption in vitro

Calcium phosphate biomaterials such as calcium deficient apatite (CDA) have been contemplated as carrier for delivery of bisphosphonate in bone tissues. In the present work, we have investigated the in vitro biological properties of Zoledronate-loaded CDA. CDA was loaded with zoledronate according to a previously described coating process. 31P MAS NMR spectra demonstrated the effective loading of zoledronate onto CDA. Using 14C labeled zoledronate, we then demonstrated the in vitro release of zoledronate from CDA. In a first set of experiments, we confirmed that Zoledronate reduced the number of TRAP-, vitronectin receptor-, and F-actin ring-positive cells as well as the resorption activity of osteoclasts obtained from a total rabbit bone cell culture. Interestingly, Zoledronate-loaded CDA and its extractive solutions decreased the osteoclastic resorption. Finally, zoledronate-loaded CDA did not affect the viability and alkaline phosphatase activity of primary osteoblastic cells. These data demonstrate that CDA is effective for loading and release of zoledronate. The released zoledronate inhibited osteoclastic resorption without affecting osteoblasts. Our findings therefore suggest that such a drug delivery system would allow an increase in the efficiency of bisphosphonates by being locally available. Further experiments are now required to evaluate the in vivo antiresorptive activity of this concept.

Characterization of antiseptic apatite powders prepared at biomimetics temperature and pH

Antiseptic apatite-based calcium phosphates were prepared as the single-phase powders. Phosphocalcic oxygenated apatites were synthesized from calcium salts and orthophosphate dissolved in oxygenated water solution at 30%, under the biomimetic conditions of 37 °C and pH 7.4. The characterization and chemical analysis of the synthesized biomimetic apatite powders were performed by scanning electron microscopy (SEM), powder X ray diffraction (XRD), Fourier-transformed infrared spectroscopy (FT-IR) and chemical analysis. The obtained materials are a calcium deficient apatites with different morphologies.