Formation Of Calcium Phosphate Layer Research Articles

Mesoporous bioactive scaffolds based on the 14.6Li2O⋅8.6ZrO2⋅67.3SiO2⋅9.5Al2O3 glass-ceramic as drug delivery for bone regeneration

Bioactive scaffolds based on the 14.6Li2O⋅8.6ZrO2⋅67.3SiO2⋅9.5Al2O3 glass-ceramic for use in drug delivery is presented. Powders of the parent glass (212 mm > particle size >38 mm) were heat-treated at 725 °C for 3 h for surface crystallization. They were subjected to a chemical attack with 6 vol% of hydrofluoric acid for 40 s for partial dissolution of the residual glass, and washed with deionised water and calcium carbonate. Structural and microstructural analyses were performed to identify the formed crystalline phases and porosity. Mesopores with 2.05 nm-radius were detected. The immersion method was used to insert alendronate sodium into the scaffolds. The drug delivering capability was evaluated by infrared spectroscopy and thermogravimetric analysis (TG). The FTIR spectra showed the typical functional groups existing in the alendronate sodium, confirming their impregnation into the scaffold's pores. Moreover, the TG thermograms showed loss weights of ∼2% indicating the effective inoculation of the drug into the scaffold. The bioactivity of the material was evaluated through calcium phosphate layer formation after incubation in a simulated body fluid for up to 28 days (37 ± 1 °C) and then analysed by infrared spectroscopy (FTIR). Calcium phosphates were identified after 24 h of immersion indicating the bioactivity of the glass-ceramic. The cytotoxicity and hemocompatibility tests showed that the LZSA glass-ceramic scaffold is not considered toxic to the human body at concentrations below 40 μg/mL and does not show hemolytic effects at all tested concentrations. Thus, the obtained microstructure renders the material promising for use as a drug delivery agent for bone regeneration.

Low-temperature bioresorbable composite material magnesium-hydroxyapatit

This work is focused on the development of «Magnesium (Mg) – Hydroxyapatite (HA)” composite for bioresorbable implant obtained by spark plasma sintering method (SPS). The structural-phase state of the samples was studied using the X-ray phase analysis method and scanning microscopy (SEM). It is essential to understand the mechanical behaviour of these materials: compressive strength were determined for material characterization. The obtained composite materials were investigated in the solution simulating the extracellular fluid of the human body (SBF) and NaCl solution, and there was shown the allocation of ions of calcium and magnesium and the formation of calcium-phosphate layer (CFL) on the surface of sintered samples and demonstrated. It follow that the material Mg–HA has a biocompatibility in vitro.

P97: Bioresorbable composites based on hydroxyapatite dispersed in poly-L-lactide matrix

Orthopedic oncologic prostheses are essential in postoperative therapy and improvement of life quality of oncological patients. However, construction and composition of implants has to be improved for better biocompatibility and reduction of inflammatory responses. The materials based on polylactic acid (PLA) and hydroxyapatite (HA) have been suggested as promising solution for implant coating in order to improve reconstruction of bone defects, to reduce risk of inflammation and implant rejection. Objective To obtain the bioresorbable composites using biocompatible poly-L-lactide (PLA) with HA and to evaluate its effect on bone remodeling rate. Materials and methods After dissolution of PLA∗ in chloroform the powder-like HA (d = 20–40 nm) was added under vigorous stirring (PLA: HA = 75:25). The PLA/HA suspension was sonicated (40 kHz) and precipitated in ethanol. Fine fibers (d = 0.1– 0.5 μm, of more than 2 mm length) were formed. The PLA/HA composite fibers were air-dried; they consisted of HA nanoparticles homogeneously distributed in the PLA matrix. The composites ability to participate in bone remodeling was estimated by determination of the polymer matrix dissolution rate and by formation of the CPL on the surface of the composites. Results The degradation rate of the polymer matrix was calculated by measuring concentration of lactic acid (by HPLC method), being released from PLA macromolecules as the result of the hydrolysis in the physiologic solution (pH 7, ω(NaCl) = 0.9%). The PLA degradation rate during the first 4 days of soaking of the substrates (d = 10 mm, S = 190 mm2, m = 0.20 ± 0.01 g) in the solution is rather high (Clactic acid = 10 wt%), while on day 5 it decreases and stays constant during the rest of the time. The HA content increase in the composite results in the enhanced hydrolysis of PLA, caused both by the partial acid-base interaction between PLA and HA, and due to presence of the phase boundaries. Interesting to note that pure PLA (without HA) is hardly hydrolyzed in the physiologic solution even on the 30-th day of the experiment. Formation of calcium phosphate layer (CPL) was done in the SBF media, simulating the mineral constituents of the blood plasma at 37 °C, over 28 days period. The total concentration of Ca2+ and Mg2+ ions in the SBF-solution was determined by daily trilonometry in the ammonia buffer with eriochrome black T as indicator. This investigation clearly shows that the substrates both of HA and PLA/HA promote the CLP formation on their surfaces. On day 14 of the substrates soaking in the SBF solution the increment of Ca2+, Mg2+ ions adsorbed on the HA substrate rose to the value of m (Ca2+ + Mg2+)/VSBF = 0.11 mg/l, while the same value on the composite PLA/HA substrate was 0.06 mg/l. Thus the rate of adsorption of Ca2+ ions on the surface of HA is higher than that on the surface of the PLA/HA substrates. It is the result of the high HA content in the material. Pure PLA does not promote adsorption of Ca2+ and Mg2+ ions from the SBF media. Thus the HA particles incorporated in the PLA matrix may cause fast the CLP formation improving the ossification in the bone reconstruction processes. Biocompatibility and anti-inflammatory activity of PLA, HA and PLA/HA composite was studied in a pilot experiment in the cell-mediated immune response of individual donors in vitro using CD14+ human monocytes. Insignificant pro-inflammatory cytokine secretion of the M1 type (TNFα) is observed in the presence of PLA, while HA and PLA/HA composites don’t contribute to their release. The PLA promotes the release of M2 cytokine CCL18 on day 6 of macrophage culturing suggesting potential anti-inflammatory properties of the material. Conclusion The composite PLA/HA supports active bone-like layer formation on its surface improving ossification in the bone reconstruction process. The pilot experiment suggested that PLA/HA does not cause inflammatory reactions, while PLA can potentially regulate the balance between M1/M2 reaction to suppress unnecessary inflammation. ∗Mol. mass ca 60 kDa, prepared in the Laboratory of Catalytic Research of Tomsk State University. This Research is supported by Tomsk State University Competitiveness Improvement Program . The work was partially supported by the project Russian Foundation for Basic Research RFBR # 15-08-05496_a and EU IMMODGEL project, Grant No. (602694). Work was conducted with the application of the Tomsk regional common use center technical equipment acquired thanks to a grant of the Russian Ministry of the Agreement No. 14.594.21.0001 (RFMEFI59414X0001).

Bioactivity and Physico-Chemical Properties of Composites on Basis of Hydroxyapatite with Lactic and Glycolic Acids Oligomers

A new methodin situfor producing of biocompatible composites based on hydroxyapatite and oligomers of lactic and glycolic acid is described. Their thermo-mechanical, physical-chemical and biological properties are studied as well. The formation of bonds between hydroxyapatite with lactic and glycolic oligomers in composites are confirmed by IR spectroscopy of the samples, including those obtained in the Soxhlet apparatus. The formation of calcium-phosphate layer on the composite's surface containing hydroxyapatite was shown by SBF-investigation. The scaffolds based on lactic acid oligomer do not stimulate formation of a calcium-phosphate layer and they are subjected to destruction by the way of hydrolysis.

Formation of hydroxyapatite layer on graphite sheet immersed in calcium phosphate solution by microwave heating

The conditions for the formation of hydroxyapatite [Ca10(PO4)6(OH)2; HAp] layer on the graphite sheet were examined via the processes of (i) the acid treatment of conc. nitric and sulfuric acids at 50°C for 24 h for the formation of carboxyl group, and (ii) the external heating (90°C, 1 h) and/or microwave heating (100°C, 5 min) of the acid-treated graphite sheet in the “apatite-dissolved solution” prepared by bubbling CO2 gas into the HAp-dispersed water. The combination of external and microwave heating was effective in the formation of large amount of calcium phosphates, because the seed crystals of apatite formed by the evaporation of solvent from the apatite-dissolved solution (i.e., external heating) contributed to enhancing the formation of calcium phosphate layer through the microwave heating. The calcium phosphate layer formed by stacking the plate-like particles possessed the typical thickness of 4.9 µm, and was identified as HAp with Ca/P ratio of 1.72 or carbonate-containing HAp. Based upon the graphite sheet with HAp layer implanted into femur and tibia of the Japanese white rabbit for 4 months, the formation of calcified bone at the interfaces was found to show the excellent biocompatibility.

Biomimetic calcium phosphate coating on Ti-7.5Mo alloy for dental application

Titanium and its alloys have been used as bone-replacement implants due to their excellent corrosion resistance and biocompatibility. However, a titanium coating is a bioinert material and cannot bond chemically to bone tissue. The objective of this work was to evaluate the influence of alkaline treatment and heat treatment on the formation of calcium phosphate layer on the surface of a Ti-7.5Mo alloy after soaking in simulated body fluid (SBF). Thirty six titanium alloy plates were assigned into two groups. For group I, samples were immersed in a 5.0-M NaOH aqueous solution at 80°C for 72 h, washed with distilled water and dried at 40°C for 24 h. For group II, after the alkaline treatment, samples were heat-treated at 600°C for 1 h in an electrical furnace in air. Then, all samples were immersed in SBF for 7 or 14 days to allow the formation of a calcium phosphate coating on the surface. The surfaces were characterized using SEM, EDS, AFM and contact angle measurements.

In vitro studies of hydrogen peroxide treated titanium for biomedical applications

Pure titanium substrate was subjected to chemical treatment with different concentrations of hydrogen peroxide and subsequently heat treated to produce a titania gel layer with anatase nature. The surface modified substrates were then immersed in simulated body fluid for the formation of calcium phosphate layer over the surface. Titanium treated with 15 wt.% of hydrogen peroxide produced a uniform porous layer, which was found to induce the calcium phosphate formation more easily. However, titanium treated with 5 wt.% and 25 wt.% of hydrogen peroxide exhibited inhomogeneous surface for the growth of calcium phosphate layer. Further, the corrosion behaviour of the untreated and hydrogen peroxide treated specimens in simulated body fluid was evaluated using potentiodynamic polarization and electrochemical impedance spectroscopy. The results have shown that the surface modified titanium using 15 wt.% of hydrogen peroxide exhibited higher corrosion resistance compared to specimens treated with other concentrations of hydrogen peroxide.

Formation of Calcium Phosphate Layer on Bioabsorbable Magnesium to Improve its Corrosion Resistance

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Bioactive and osteoblast cell attachment studies of novel α- and β-chitin membranes for tissue-engineering applications

Chitin is a novel biopolymer and has excellent biological properties such as biodegradation in the human body and biocompatible, bioabsorable, antibacterial and wound healing activities. In this work, α- and β-chitin membranes were prepared using α- and β-chitin hydrogel. The bioactivity studies were carried out using these chitin membranes with the simulated body fluid solution (SBF) for 7, 14 and 21 days. After 7, 14 and 21 days the membranes were characterized using SEM, EDS and FT-IR. The SEM, EDS and FT-IR studies confirmed the formation of calcium phosphate layer on the surface of the both chitin membranes. These results indicate that the prepared chitin membranes were bioactive. Cell adhesion studies were also carried out using MG-63 osteoblast-like cells. The cells were adhered and spread over the membrane after 24 h of incubation. These results indicated that the chitin membranes could be used for tissue-engineering applications.

Preparation, characterization, bioactive and cell attachment studies of α-chitin/gelatin composite membranes

The chitin/gelatin composite membranes were prepared by mixing of chitin hydrogel with gelatin. The prepared composite membranes were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), mechanical, swelling, enzymatic degradation and thermal studies. The XRD pattern of the chitin/gelatin composite membranes showed almost the same pattern as α-chitin. The bioactivity studies of these chitin/gelatin membranes were carried out with the simulated body fluid solution (SBF) for 7, 14 and 21 days followed by the characterization with the scanning electron microscopy (SEM) and Energy Dispersive Spectrum (EDS) studies. The SEM and EDS studies confirmed the formation of calcium phosphate layer on the surface of chitin/gelatin membranes. Biocompatibility of the chitin/gelatin membrane was assessed using human MG-63 osteoblast-like cells. After 48 h of incubation, it was found that the cells had attached and completely covered the membrane surface. Thus, the prepared chitin/gelatin membranes are bioactive and are suitable for cell adhesion suggesting that these membranes can be used for tissue-engineering applications.

Hydroxyapatite Nucleation on Modified Silicon Surface for Use in Biosensors

In this work, we explore the deposition of single-phase and uniform HA coatings on a crystalline silicon surface of microelectronic quality. The used methodology includes an NaOH treatment to produce the silanization of Si surface and a modified biomimetic technique, using sodium silicate solution (SS) as nucleant agent, for apatite-based coating formation. The sodium silicate solution (SS) demonstrated to be effective for the calcium phosphate layer formation, but the treatment can reduce the induction to obtain a uniform surface coating of HA. This suggests that, among the factors that determine nucleation of calcium phosphate on silicon, surface electrical charge could play a key role.

Formation of calcium phosphate layer on ceramics with different reactivities

Biphasic ceramic samples of different biological reactivity are prepared by using hydroxyapatite (HAp) and tricalcium phosphate (TCP) in various ratios. Different parameters for sintering in an air atmosphere furnace were defined after dilatometric studies. An increased densification with decreased TCP content was observed. The sintered bodies were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The dissolution kinetics and in vitro reactivity were investigated using simulated body fluid (SBF) at 37 °C for a maximum period of 3 weeks. The surfaces of the ceramics were analyzed by Fourier transform infrared spectroscopy (FTIR) and SEM in order to observe the formation of a calcium phosphate layer, which indicates the samples bioactivity. Dissolution in SBF demonstrated that layers with different kinetics on the samples surface were formed during the immersion period. The biphasic ceramics show bioactive behavior, even if the resorbable TCP is incorporated.

The effect of bioactive glass content on synthesis and bioactivity of composite poly (lactic-co-glycolic acid)/bioactive glass substrate for tissue engineering

Tissue engineering offers a promising new approach to bone tissue grafting. One material that has received attention in this regard is the polymer poly (lactic-co-glycolic acid) (PLGA). It has the advantage of controllable bioresorption and ease of processing. Another material of interest is bioactive glass (BG), which shows the ability to stimulate osteoblastic differentiation of osteoprogenitor cells. In this study, we reported on the optimal synthesis parameters and the kinetics of formation of calcium phosphate (Ca-P) phase at the surface of PLGA/BG composites. The formation of calcium phosphate layer was confirmed using scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDXA). PLGA-30%BG microspheres based porous scaffolds for bone tissue engineering were examined for their ability to promote osteogenesis of marrow stromal cells (MSC). This porous scaffold supported both MSC proliferation and promoted MSC differentiation into cells expressing the osteoblast phenotype. It therefore demonstrates significant potential as a bone replacement material.

Characterization of microrough bioactive glass surface: surface reactions and osteoblast responses in vitro.

The current study characterized the in vitro surface reactions of microroughened bioactive glasses and compared osteoblast cell responses between smooth and microrough surfaces. Three different bioactive glass compositions were used and surface microroughening was obtained using a novel chemical etching method. Porous bioactive glass specimens made of sintered microspheres were immersed in simulated body fluid (SBF) or Tris solutions for 1, 6, 24, 48, or 72 h, and the formation of reaction layers was studied by means of a scanning electron microscope/energy dispersive X-ray analysis (SEM/EDXA). Cell culture studies were performed on bioactive glass disks to examine the influence of surface microroughness on the attachment and proliferation of human osteoblast-like cells (MG-63). Cell attachment was evaluated by means of microscopic counting of in situ stained cells. Cell proliferation was analyzed with a nonradioactive cell proliferation assay combined with in situ staining and laser confocal microscopy. The microroughening of the bioactive glass surface increased the rate of the silica gel layer formation during the first hours of the immersion. The formation of calcium phosphate layer was equal between control and microroughened glass surfaces. In cell cultures on bioactive glass, the microrough surface enhanced the attachment of osteoblast-like cells but did not have an effect on the proliferation rate or morphology of the cells as compared with smooth glass surface. In conclusion, microroughening significantly accelerated the early formation of surface reactions on three bioactive glasses and had a positive effect on initial cell attachment.

Effect of poling conditions on growth of calcium phosphate crystal in ferroelectric BaTiO3 ceramics.

Recently, ceramic materials have been given a lot of attention as candidates for implant materials, since they possess biologically favorable characteristics for osseointegration. Among them, BaTiO3 (BTO) ceramics are ferroelectric and piezoelectric after poling treatments. However, little or no information is available on the poling condition of BTO and their effect on calcium phosphate (CaP) formation. In this study, the effect of poling conditions on the formation of CaP layer was investigated. It was observed from this study that CaP was formed on negatively charged BTO surfaces. An increase in Ca/P ratio to 1.67 was observed when the poling temperature was increased above the Curie temperature. On positively charged BTO, no CaP layer was observed.

Effect of the substitution of M 2O 3 (M=La, Y, In, Ga, Al) for CaO on the bioactivity of 2.5CaO·2SiO 2 glass

Glasses were prepared whose compositions are defined by the following general formula: (2.5− x)CaO· x/3M 2O 3·2SiO 2 (0⩽ x⩽0.6) (M=Ga, Al, In). Their bioactivity was studied “in vitro” by soaking the glasses in a simulated body fluid (SBF) The consequent formation of calcium phosphate layer was studied by means of electron microscopy (EM) equipped with an energy-dispersive system (EDS) for elemental analysis and Infrared (IR) spectroscopy. The results are compared to the literature relative to the substitution of La 2O 3 and Y 2O 3 for CaO. It is observed that, in general, the substitution of M 2O 3 for CaO in the binary CaO–SiO 2 glass composition progressively reduces the ability to form a calcium phosphate layer on the surfaces exposed to simulated body fluid (SBF). The composition limit can be related to the ionic field strength of the substituting cation and to the CaO content of the base glass. According to the mechanism reported in the literature a silica gel-like surface layer initially forms on the surfaces exposed to SBF. The observed results can be attributed to the effect of the substitution of M 2O 3 for CaO on the acidic properties of the silanolic groups.

45S5 Bioactive glass surface charge variations and the formation of a surface calcium phosphate layer in a solution containing fibronectin

This study investigated the effect of fibronectin adsorption on surface charge variations and calcium phosphate (Ca-P) layer formation kinetics on the surface of 45S5 bioactive glass (BG). We hypothesize that the adsorption of fibronectin on BG changes the surface charge and alters the kinetics of Ca-P layer formation on the glass surface. The charge at a material's surface modulates surface chemistry, protein adsorption, and interactions with bone cells. The zeta potential of BG in a solution containing human plasma fibronectin (TE-FN) was measured as a function of time by particle electrophoresis, and Ca-P layer formation was characterized using SEM, EDXA, and FTIR. Si, Ca, and P solution concentrations also were determined. It was found that the adsorption of fibronectin reduced the initial electronegativity of the BG surface and delayed the formation of both the amorphous and the crystalline Ca-P layers. The delayed formation of these surface layers may be attributed to the competitive binding of Ca2+ ions by the fibronectin molecule. In addition, the formation of an amorphous Ca-P layer correlated with the reversal from a negatively to a positively charged surface, independent of the presence of fibronectin. The addition of a single protein (in this case fibronectin) can significantly alter material surface parameters, such as charge, and subsequently affect the formation of a surface Ca-P layer. Furthermore, the formation of an amorphous Ca-P layer is an important event in the reactions leading to bioactive behavior, and proteins such as FN are actively involved in the transformation of the surface into a Ca-P layer. © 2000 Wiley & Sons, Inc. J Biomed Mater Res 54: 454–461, 2001

Temporal zeta potential variations of 45S5 bioactive glass immersed in an electrolyte solution

45S5 bioactive glass (BG) is a bioactive material known to bond to bone in vivo through a surface calcium phosphate (Ca-P) layer. The goal of this study was to address the importance of BG surface charge in the bioactive response by examining the relationship between charge variations and the formation of the surface Ca-P layer. The zeta potential of BG in an electrolyte solution (TE) was measured by particle electrophoresis, and the formation of a Ca-P layer was characterized using SEM, EDXA, and FTIR. Si, Ca, and P solution concentrations also were determined. The initial BG surface was negatively charged, and two sign reversals were detected during 3 days of immersion. The first, from negative to positive after 1 day, is attributed to the adsorption of cations at the BG surface, and the second reversal was due to the precipitation of phosphate ions from solution. A strong correlation was found between the formation of a Ca-P layer and BG surface zeta potential variations. The dynamic shift in zeta potential from an initially negative surface to a positively charged surface directly corresponded with the formation of an amorphous Ca-P layer. In addition, when the glass surface matured into a crystalline Ca-P layer, it was associated with a reversal from a positive to a negative surface. Future work will focus on the effects of protein adsorption on BG surface charge and Ca-P layer formation kinetics as well as on cellular response to a changing BG surface.

45S5 bioactive glass surface charge variations and the formation of a surface calcium phosphate layer in a solution containing fibronectin.

This study investigated the effect of fibronectin adsorption on surface charge variations and calcium phosphate (Ca-P) layer formation kinetics on the surface of 45S5 bioactive glass (BG). We hypothesize that the adsorption of fibronectin on BG changes the surface charge and alters the kinetics of Ca-P layer formation on the glass surface. The charge at a material's surface modulates surface chemistry, protein adsorption, and interactions with bone cells. The zeta potential of BG in a solution containing human plasma fibronectin (TE-FN) was measured as a function of time by particle electrophoresis, and Ca-P layer formation was characterized using SEM, EDXA, and FTIR. Si, Ca, and P solution concentrations also were determined. It was found that the adsorption of fibronectin reduced the initial electronegativity of the BG surface and delayed the formation of both the amorphous and the crystalline Ca-P layers. The delayed formation of these surface layers may be attributed to the competitive binding of Ca2+ ions by the fibronectin molecule. In addition, the formation of an amorphous Ca-P layer correlated with the reversal from a negatively to a positively charged surface, independent of the presence of fibronectin. The addition of a single protein (in this case fibronectin) can significantly alter material surface parameters, such as charge, and subsequently affect the formation of a surface Ca-P layer. Furthermore, the formation of an amorphous Ca-P layer is an important event in the reactions leading to bioactive behavior, and proteins such as FN are actively involved in the transformation of the surface into a Ca-P layer.