Formation Of Bone-like Hydroxyapatite Research Articles

Sol-gel synthesis and characterization of novel cobalt ions-containing mesoporous bioactive glass nanospheres as hypoxia and ferroptosis-inducing nanotherapeutics

Herein, novel Co2+ ions-containing mesoporous bioactive glass nanospheres (Co-BGn) with low and high cobalt contents were prepared by a facile one-pot ultrasonic-coupled sol-gel synthesis. Structural evaluations revealed that Co-BGn are totally amorphous and cobalt ions exist as Co2+ ions in the glass structure. Notably, Co-BGn exhibited uniform spherical morphology, nanosize (< 100 nm), high worm-like mesoporosity (0.41 cm3/g) and large specific surface area ca. 787.5 m2/g. Interestingly, low and high Co-BGn showed sustained release of Co2+ ions with controlled release (8 – 32 ppm) and fast release (54 – 112.5 ppm) kinetics over 2 weeks, respectively. Furthermore, Co-BGn showed good bone-like hydroxyapatite formation in vitro. Accordingly, low Co-BGn with controlled release of Co2+ ions can be considered as hypoxia-mimic nanotherapeutics for bone/skin tissue regeneration. Whereas, high Co-BGn with fast release of high concentrations of Co2+ ions can be considered for ferroptosis killing of cancer cells including bone and skin cancers.

Antioxidant cerium ions-containing mesoporous bioactive glass ultrasmall nanoparticles: Structural, physico-chemical, catalase-mimic and biological properties

The multifunctional biological properties of Ce ions including antioxidant, anti-inflammatory, antibacterial and anti-cancer effects are very encouraging for development of Ce-containing biomaterials with therapeutic properties. Herein, novel Ce3+/Ce4+ ions containing mesoporous bioactive glass ultrasmall nanoparticles (Ce-BGn) were prepared by a facile one-pot ultrasound-assisted sol-gel method. Interestingly, Ce2O3 incorporation exerted a significant influence on the particle size and textural properties of mesoporous BGn (SiO2 – CaO binary glass system). Ce-BGn exhibited ultrasmall nanoparticle size (< 30 nm), mesoporous texture (pore size up to 2.82 nm and pore volume up to 0.191 cm3/g) and large specific surface area ca. 132.9 m2/g. Notably, in situ formation of CeO2 nanospheres (3–6 nm) was detected at the surface and in the amorphous glass matrix of mesoporous Ce-BGn. Importantly, X-ray photoelectron spectroscopy (XPS) revealed the presence of 72.57 % Ce3+ and 27.43 % Ce4+ at the surface of mesoporous Ce-BGn with Ce3+/Ce4+ ratio = 2.66. Furthermore, mesoporous Ce-BGn exhibited high catalase-mimic activity and showed sustained release of Ce (2.5–32 ppm), Ca (85–327 ppm) and Si (54–200 ppm) ions within 4 weeks along with excellent bone-like hydroxyapatite formation. Finally, the in vitro biological behavior of mesoporous Ce-BGn in cell cultures of human skin fibroblasts (HSF) revealed that mesoporous Ce-BGn (with concentrations up to 300 μg/mL) possess good cyto-biocompatibility. Taken together, novel ultrasmall mesoporous Ce-BGn showed remarkable catalase-mimic activity via surface containing Ce3+/Ce4+ ions which can scavenge ROS (Ce3+↔ Ce4+) and decompose H2O2 molecules into H2O and O2. In addition to that, Ce-BGn demonstrated sustained release of bioactive ions (Ce, Ca and Si), excellent bone-like hydroxyapatite formation and good cyto-biocompatibility.

Bioactivity reinforced surface patch bound collagen-pectin hydrogel

In this report, we discuss the design of a novel collagen/pectin (CP) hybrid composite hydrogel (CPBG) containing in-situ mineralized bioactive glass (BG) particles to simulate an integrative 3D cell environment. Systematic analysis of the CP sol revealed collagen and pectin molecules interacted regardless of both possessing similar net negative charge through the mechanism of surface patch binding interaction. Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) confirmed this associative interaction which resulted in the formation of a hybrid crosslinked network with the BG nanoparticles acting as pseudo crosslink junctions. Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray Analysis (EDAX) and Transmission Electron Microscopy (TEM) results confirmed uniform mineralization of BG particles, and their synergetic interaction with the network. The in-vitro bioactivity tests on CPBG indicated the formation of bone-like hydroxyapatite (Ca10(PO4)6(OH)2) microcrystals on its surface after interaction with simulated body fluid. This hydrogel was loaded with a model antifungal drug amphotericin-B (AmB) and tested against Candida albicans. The AmB release kinetics from the hydrogel followed the Fickian mechanism and showed direct proportionality to gel swelling behavior. Rheological analysis revealed the viscoelastic compatibility of CPBG for the mechanical load bearing applications. Cell viability tests indicated appreciable compatibility of the hydrogel against U2OS and HaCaT cell lines. FDA/PI on the hydrogel portrayed preferential U2OS cell adhesion on hydrophobic hydroxyapatite layer compared to hydrophilic surfaces, thereby promising the regeneration of both soft and hard tissues.

The Effect of Material Surface Microstructure on the Enhancement of Bone-Bonding Ability of a Hydroxyapatite-Implant by Low-Intensity Pulsed Ultrasound (LIPUS)

Excellent firm bonding between the biomaterials and bone tissue (osseointegration and osteo-conductivity) has been desired for the stability in vivo of dental implants and artificial joints. Much has been learned about this concept, which has led to significant improvements in the design and surface modification of implants in the field of implant dentistry, orthopedic surgery. We have already reported that low-intensity pulsed ultrasound (LIPUS) irradiation can accelerate the bone bonding ability of the bio-conductive materials such as bioactive titanium and hydroxyapatite implant. However, it is still unclear whether the LIPUS could have same effect to different types of the bioactive-materials. Therefore, in this study, the differences of bone-like hydroxyapatite formation on some kind of hydroxyapatite surface in simulated body fluid (SBF) under the LIPUS irradiation were investigated. Two kinds of hydroxyapatite samples immersed in SBF was exposed to ultrasound waves, the bone-like apatite on the surface was analyzed by Scanning electron microscopy and X-ray diffraction. As a result, the enhancement of hydroxyapatite formation on the surface by LIPUS was confirmed, the initial epitaxial nucleation and crystal growth of apatite depended on crystal structure of the surface of matrix materials.

Hydroxyl Groups Induce Bioactivity in Silica/Chitosan Aerogels Designed for Bone Tissue Engineering. In Vitro Model for the Assessment of Osteoblasts Behavior.

Silica (SiO2)/chitosan (CS) composite aerogels are bioactive when they are submerged in simulated body fluid (SBF), causing the formation of bone-like hydroxyapatite (HAp) layer. Silica-based hybrid aerogels improve the elastic behavior, and the combined CS modifies the network entanglement as a crosslinking biopolymer. Tetraethoxysilane (TEOS)/CS is used as network precursors by employing a sol-gel method assisted with high power ultrasound (600 W). Upon gelation and aging, gels are dried in supercritical CO2 to obtain monoliths. Thermograms provide information about the condensation of the remaining hydroxyl groups (400–700 °C). This step permits the evaluation of the hydroxyl group’s content of 2 to 5 OH nm−2. The formed Si-OH groups act as the inductor of apatite crystal nucleation in SBF. The N2 physisorption isotherms show a hysteresis loop of type H3, characteristic to good interconnected porosity, which facilitates both the bioactivity and the adhesion of osteoblasts cells. After two weeks of immersion in SBF, a layer of HAp microcrystals develops on the surface with a stoichiometric Ca/P molar ratio of 1.67 with spherulite morphology and uniform sizes of 6 μm. This fact asserts the bioactive behavior of these hybrid aerogels. Osteoblasts are cultured on the selected samples and immunolabeled for cytoskeletal and focal adhesion expression related to scaffold nanostructure and composition. The initial osteoconductive response observes points to a great potential of tissue engineering for the designed composite aerogels.

Mesoporous electroactive silver doped calcium borosilicates: Structural, antibacterial and myogenic potential relationship of improved bio-ceramics

In this work improved electroactive mesoporous Ag-doped bio-ceramics for medical usages are developed, examining their structural, electrical, in-vitro bioactivity, cell cultures and antibacterial properties against various classical pathogenic bacteria. Ag-containing mesoporous bio-ceramics (MBCs): xmol%Ag2O - (100-x)[45.8CaO-8.4B2O3-45.8SiO2] where x = 2, 5, 7.5 and 10 were synthesized through a sol-gel method. The small angle X-ray scattering and electron microscopy studies reveal the embedment of silver nanoparticles in the samples. Existence of silver as Ag+/Ag0 forms in the samples is confirmed by X-ray photoelectron spectroscopy. The N2 adsorption-desorption analysis evidence the mesoporous structure of the samples. The electrical conductivity of samples increases from 5.4 x 10−8 S cm−1 for x = 2 to 1.9 x 10−6 S cm−1 for x = 7.5 and then decreases to 0.9 x 10−6 S cm−1 for x = 10 at 110 °C. In vitro bioactivity studies revealed that Ag-containing MBCs hold the bone-like hydroxyapatite formation after immersion in human blood plasma like-solution such as Dulbecco's Modified Eagle's Medium. The antibacterial effect of samples against pathogenic bacteria (S. aureus, E. coli, P. monas aeruginosa, and B. cereus) increases with Ag concentration (x = 7.5) and then decreases with Ag content (x = 10). Antibacterial effect is greater for the sample with high electrical conductivity. The cell culture studies evidence not considerable cytotoxic effects for Ag-containing MBCs. Finally, the C2C12 myoblast cell culture studies reveal the significant cell growths and differentiation (myogenesis) for high electrical conducting Ag-containing MBCs.

Biocorrosion and biodegradation behavior of ultralight Mg–4Li–1Ca (LC41) alloy in simulated body fluid for degradable implant applications

Biocorrosion and biodegradation behavior of Mg–4Li–1Ca alloy were investigated for industrially important end product conditions, namely the homogenized, rolled, and rolled + annealed ones. Among the three, homogenized material showed the highest corrosion rate (27.2 mm/year) in a simulated body fluid (SBF) owing to its coarse grain structure containing long dumbbell-shaped eutectic phase. Rolled + annealed material exhibited the lowest corrosion rate (0.94 mm/year) corresponding to the highest corrosion resistance (1.854 kΩ cm2) in SBF. This higher corrosion resistance is associated with a uniform distribution of corrosion sites and a lower occurrence of twins in the microstructure. However, the rolled material showed a greater corrosion rate due to an appreciable volume fraction of {10\( \bar{1} \)1} compression twins, {10\( \bar{1} \)2} tension twins, and {10\( \bar{1} \)1}–{10\( \bar{1} \)2} double twins, which form galvanic couples with the adjacent grains that enhances localized corrosion. A mechanism of biodegradation at the alloy/SBF interface is proposed. It involves the formation of bone-like hydroxyapatite and metastable octa calcium phosphate, along with other degradation products, such as magnesium hydroxide and lithium hydroxide.

Mineralization of hydroxyapatite crystallites on zein microspheres

AbstractBioinspired mineralization process has been extended to the formation of bonelike hydroxyapatite (HAp) coatings on biodegradable polymer substrates. HAp‐coated zein microspheres were prepared through phase separation procedure, followed by incubation in modified simulated body fluids (mSBF). The morphologies of mineralized zein microspheres with different diameters and incubation time were investigated by scanning electron microscopy (SEM). The nature of the deposited mineral phase was characterized by energy‐dispersive spectroscopy (EDS) and wide‐angle X‐ray diffraction (XRD). The results showed that as the average diameter of zein microspheres increased to 1.13 μm, randomly oriented minerals were observed on the surface of microspheres. Two characteristic HAp diffraction reflections 002 and 211 centered at 2θ of 25.6° and 32.0° were ascertained on the XRD patterns of the mineralized microspheres. Incubation of zein microspheres with larger diameters in mSBF led to nucleation and growth of minerals on the surface. Even larger number of minerals were deposited and a porous structure of platelike HAp crystals was obtained after incubation for 21 days. The novel zein/HAp microspheres may have a wide variety of potential applications in bone regeneration and tissue engineering. POLYM. COMPOS., 2012. © 2012 Society of Plastics Engineers

Calcium phosphate growth at electropolished titanium surfaces.

This work investigated the ability of electropolished Ti surface to induce Hydroxyapatite (HA) nucleation and growth in vitro via a biomimetic method in Simulated Body Fluid (SBF). The HA induction ability of Ti surface upon electropolishing was compared to that of Ti substrates modified with common chemical methods including alkali, acidic and hydrogen peroxide treatments. Our results revealed the excellent ability of electropolished Ti surfaces in inducing the formation of bone-like HA at the Ti/SBF interface. The chemical composition, crystallinity and thickness of the HA coating obtained on the electropolished Ti surface was found to be comparable to that achieved on the surface of alkali treated Ti substrate, one of the most effective and popular chemical treatments. The surface characteristics of electropolished Ti contributing to HA growth were discussed thoroughly.

Low temperature, pH-triggered synthesis of collagen–chitosan–hydroxyapatite nanocomposites as potential bone grafting substitutes

A pH-triggered synthesis strategy at low temperature was developed to initiate collagen self-assembly, chitosan precipitation and hydroxyapatite synthesis by ammonia diffusion into the acidic homogeneous stock solution for preparing bone-like nanocomposites. FTIR and XRD analyses were used to confirm the molecular interactions and the formation of bone-like hydroxyapatite in collagen–chitosan–hydroxyapatite nanocomposites. A facile modulation of hydroxyapatite content in the obtained nanocomposites was further validated by thermogravimetric analysis. SEM observations revealed that the unique microstructure of obtained nanocomposites was composed of collagen fibrils and nano needle-like objects dependent upon the relative ratio of inorganic to organic components. This strategy is convenient and also feasible to prepare collagen based bone-like nanocomposites.

Preliminary in vitro Study on Enhancement of Bone-like Hydroxyapatite Formation on Bio-active Titanium Alloy by Low-intensity Pulsed Ultrasound Waving for Early Bone Bonding

In this study, we investigated whether ultrasound wave stimulation could accelerate the bone-bonding ability of bio-active titanium alloy in vitro. Titanium alloy (α+β-Ti-6Al-4V) processed in chemical and heat treatments was used as a specimen, and soaked in simulated body fluid (SBF) under pulsed ultrasound wave for the planned time periods. The surface of samples was observed using scanning electron microscopy (SEM), energy dispersive spectroscopy, X-ray diffraction to assess the state of bone-like hydroxyapatite formation. SEM images showed that a richer and finer layer of bone-like hydroxyapatite covered the titanium surface in the ultrasound wave group as compared with the non-ultrasound group. The measurements of mass of specimens also indicated the efficiency of ultrasound waves for hydroxyapatite formation. These findings suggest that the nucleation and crystallization of apatite on bio-active material surfaces might be promoted by micro-moving and cavitation of low-intensity pulsed ultrasound waves. We propose that pulsed ultrasound stimulation has a great potential for further improvement of osseointegration and osteoconductivity for medical bio-active implants.

Induced deposition of bone-like hydroxyapatite on thermally oxidized titanium substrates using a spatial gap in a solution that mimics a body fluid

We report on the effect of using a spatial gap on heterogeneous nucleation on the surface of thermally oxidized titanium substrates. Induction of heterogeneous nucleation of bone-like hydroxyapatite (BHAp) was evaluated in the spatial gaps between substrates that were thermally oxidized at temperatures of 100-800°C on exposure to a simulated body fluid (SBF). After soaking in a SBF for 7 d, BHAp spontaneously deposited inside the gap on the surface of samples that were thermally oxidized at temperatures above 400°C, but not on samples that were thermally oxidized at temperatures of 300°C or less. Among the substrates studied, BHAp particles were most readily deposited inside the gap on the surface of the samples that were thermally oxidized at 400°C after soaking in an SBF. A smaller gap led to a higher number of BHAp particles being deposited on the surface of the samples that were thermally oxidized at 400 or 500°C. Our results suggest that the formation of BHAp in a SBF is dependent on the temperature during thermal oxidization, and also on the spatial gap between the samples. The ease of formation of BHAp on thermally oxidized titanium increases with increasing thickness of the rutile phase and the number of Ti-OH groups, which are produced during the thermal oxidization process. In addition to the surface structure of the substrates, the spatial gap is regarded as an important parameter for enhancing the deposition of BHAp. Since the formation of BHAp allows osteoconduction to occur after implantation in a bony defect, it is possible to design titanium-based implants with a high biological affinity to bone by processing using an appropriate spatial design of the substrate.

Nanometer‐scale surface modification of Ti6Al4V alloy for orthopedic applications

This communication presents a novel technology to enhance the biocompatibility of bioinert Ti6Al4V alloy as implant materials for orthopaedic application. The surface of Ti6Al4V alloy was electrochemically activated in NaOH solution to create a porous structure with nanometer topographic features and an alkaline environment, thus promoting the formation of bone-like hydroxyapatite coating and enhancing the bonding strength of the coating. This innovative activation process was proved to be effective and essential. The activated surface was confirmed to be pure TiO2 and the formed coating was characterized of pure hydroxyapatite with a nanometer-scaled grain size structure by means of XPS, FESEM/SEM/EDX, XRD, and TEM techniques.

Bioactive titanium-particle-containing dicalcium silicate coating

Titanium particles with typical size ranging from 80 to 140 μm were used as scaffolds in the synthesis of bioactive dicalcium silicate (finer than 20 μm) coating by atmospheric plasma spraying to improve the physiological chemical bonding between the coating and bone. A bonding strength as high as 49.0 MPa was achieved between the coating and substrate as a result of the high titanium content in the coating and the similar thermal expansion coefficients of the coating and substrate. The favorable bioactivity was also accomplished as revealed by the formation of bone-like hydroxyapatite after immersion in simulated body fluids (SBF) for 7 days. The good biological properties were further demonstrated by adhesion and differentiation of human osteogenetic cells seeded directly on the coating surface. After dissolution experiments conducted in a Tris–HCl solution, very little changes in the mechanical properties were observed demonstrating the good durability of the coating. Our results thus show that the composite coating possesses not only good bioactivity but also long-term durability.

Local induction of calcium phosphate formation on TiO2 coatings on titanium via surface treatment with a CO2 laser.

Sol-gel-derived TiO(2) coatings are known to promote bonelike hydroxyapatite formation on their surfaces in vitro and in vivo. Hydroxyapatite integrates into bone tissue. In some clinical applications, the surface of an implant is simultaneously interfaced with soft and hard tissues, so it should match the properties of both. A new method is introduced for treating the coatings locally in a controlled manner. The local densification of sol-gel-derived titania coatings on titanium substrates with a CO(2) laser was studied in terms of the in vitro calcium phosphate-inducting properties. CO(2)-laser-treated multilayer coatings were compared with furnace-fired coatings prepared with the same recipe and previously shown to be bioactive. Additionally, local areas of furnace-fired multilayer coatings (previously shown to be bioactive in vitro) were further laser-treated to achieve various properties in the same implant. Topological surface properties were examined with atomic force microscopy. The formation of hydroxyapatite was studied with Fourier transform infrared and scanning electron microscopy energy-dispersive X-ray analysis. The results show that calcium phosphate formation can be adjusted locally by laser treatment. Calcium phosphate is a bonelike hydroxyapatite. The local treatment of sol-gel-derived coatings with a CO(2) laser is a promising technique for creating implants with various properties to interface different tissues and a possible way of coating implants that do not tolerate furnace firing.

Effect of Albumin and Fibrinogen on Calcium Phosphate Formation on Sol−Gel-Derived Titania Coatings in Vitro

The sol−gel technique provides a method to produce porous titania (TiO2) coatings, which are known to induce bone-like hydroxyapatite formation on their surface in vitro. In this study, the calcium phosphate formation (in vitro bioactivity) on a sol−gel-derived titania coating was investigated in vitro in a simulated body fluid in the presence and absence of albumin (BSA) and fibrinogen (Fib) in solution as well as the effect of surface immobilized proteins on the biomineralization process. The effect of proteins on calcium phosphate (CP) formation was followed by ion concentration analysis, XRD, SEM-EDX, and XPS. When BSA and Fib were present in solution, the CP layer growth kinetics were strongly retarded. It is suggested that the bone-like apatite formation on sol−gel-derived titania coatings occurs via continuous dissolution/reprecipitation processes, where the initially formed CP phase(s) recrystallizes into a more thermodynamically stable phase(s), as previously observed for other biomaterials. Inho...

The role of hydrated silica, titania, and alumina in inducing apatite on implants

Pure soluble silica prepared by a sol-gel method induced bone-like hydroxyapatite formation onto its surface when the silica was immersed in a simulated body fluid (SBF), whereas silica glass and quartz did not. This finding directly supports the hypothesis that hydrated silica plays an important role in biologically active hydroxyapatite formation on the surfaces of bioactive glasses and glass-ceramics, which leads to bone-bonding. Gel-derived titania is also a hydroxyapatite inducer because of its abundant TiOH groups. These results provide further insight into the unique osseointegration of titanium and its alloys. It is suspected that gel-derived titania develops an apatite layer by taking calcium and phosphate from the body fluid, thus producing bone-bonding. Although sufficient AlOH groups may remain in the alumina gel, they do not serve to initiate apatite generation when immersed in SBF. This phenomenon explains the fact that an intermediate fibrous tissue is usually found to separate the alumina implant from bone. One may infer that both abundant OH groups and negatively charged surfaces of gel-derived silica and titania are important for hydroxyapatite induction. material which possesses and/or develops both a negatively charged surface and abundant OH groups in a physiologically-related fluid is most likely to be an efficient apatite inducer. Such materials are suitable candidates to serve as bone-bonding biomaterials.