In vitro evaluation of bioactive strontium-based ceramic with rabbit adipose-derived stem cells for bone tissue regeneration

The development of bone replacement materials is an important objective in the field of orthopaedic surgery. Due to the drawbacks of treating bone defects with autografts, synthetic bone graft materials have become optional. So in this work, a bone tissue engineering approach with radiopaque bioactive strontium incorporated calcium phosphate was proposed for the preliminary cytocompatibility studies for bone substitutes. Accumulating evidence indicates that strontium containing biomaterials promote enhanced bone repair and radiopacity for easy imaging. Hence, strontium calcium phosphate (SrCaPO₄) and hydroxyapatite scaffolds have been investigated for its ability to support and sustain the growth of rabbit adipose-derived mesenchymal stem cells (RADMSCs) in vitro. They were characterized via Micro-CT for pore size distribution. Cells used were isolated from New Zealand White rabbit adipose tissue, characterized by FACS and via differentiation into the osteogenic lineage by alkaline phosphatase, Masson’s trichome, Alizarin Red and von Kossa staining on day 28. Material-cell interaction was observed by SEM imaging of cell morphology on contact with material. Live–Dead analysis was done by confocal laser scanning microscopy and cell cluster analysis via μCT. The in vitro biodegradation, elution and nucleation of apatite formation of the material was evaluated using simulated body fluid and phosphate buffered saline in static regime up to 28 days at 37 °C. These results demonstrated that SrCaPO₄ is a good candidate for bone tissue engineering applications and with osteogenically-induced RADMSCs, they may serve as potential implants for the repair of critical-sized bone defects.     

一步溶剂热法合成锶掺杂羟基磷灰石超长纳米线

羟基磷灰石超长纳米线可用于构建不同种类的生物材料, 如高柔性生物医用纸和弹性多孔骨缺损修复支架, 在生物医学领域具有良好的应用前景。锶元素作为一种微量元素, 在骨代谢过程中起着重要作用。本研究通过一步溶剂热法合成了具有不同锶掺杂量的羟基磷灰石超长纳米线; 研究了不同锶掺杂量对羟基磷灰石超长纳米线的形貌和物相的影响。所制备的锶掺杂羟基磷灰石超长纳米线具有高柔韧性和超长一维纳米结构。能量色散谱、X射线粉末衍射和傅里叶变换红外光谱分析表明, 锶元素成功地掺杂到了羟基磷灰石超长纳米线中。本研究发展的制备方法可以制备锶/(锶+钙)摩尔比从0到100%任一比例的锶掺杂羟基磷灰石超长纳米线, 大幅拓展了羟基磷灰石超长纳米线在骨缺损修复和牙科修复等生物医学领域中的应用。     

Synthesis and properties of bio-waste-based hydroxyapatite via hydrothermal process

In this study, a facile hydrothermal method is applied to produce hydroxyapatite (HA) particles using egg shells as calcium precursors and fruit waste extracts (banana peel) as biomolecular templates at 150 °C for a reaction time period of 12 hours (h) and 24 hours. The sintering of the green samples of hydroxyapatite were conducted at 1250 °C in air for 2 h. The results showed that pectin extracted from banana peel extracts assisted in regulating crystal growth to obtain homogeneous hydroxyapatite powder, with higher purity observed for 24 h hydrothermal reaction time. Fourier-transform infrared spectroscopy (FTIR) spectra revealed the presences of phosphate (PO₄³⁻) and hydroxyl (OH⁻) groups in the powders. A relative density of 89.6 % was achieved for sintered hydroxyapatite compacts produced via hydrothermal method for 24 h. The sintered body was characterized by having high Vickers hardness of 5.35 GPa and good fracture toughness of 1.23 MPa√m, suitable for biomedical application.     

Hydroxyapatite forming ability of electrostatic spray pyrolysis derived calcium phosphate nano powder

A novel fabrication technique, i.e., electrostatic spray pyrolysis (ESP), has been used in this study to prepare calcium phosphate nano powders. Final annealing was done at 400°C for 30 min in air. The hydroxyapatite-forming ability of the annealed powder has been investigated in Eagle’s minimum essential medium solution. X-ray diffracton, field emission scanning electron microscope, energy dispersive X-ray spectroscope, and Fourier transform infrared spectroscope were employed to characterize the annealed powders after immersion. The powder with an amorphous structure induced hydroxyapatite formation on their surfaces after immersion for 15 days.     

Hydroxyapatite forming ability of electrostatic spray pyrolysis derived calcium phosphate nano powder

A novel fabrication technique, i.e., electrostatic spray pyrolysis (ESP), has been used in this study to prepare calcium phosphate nano powders. Final annealing was done at 400°C for 30 min in air. The hydroxyapatite-forming ability of the annealed powder has been investigated in Eagle’s minimum essential medium solution. X-ray diffracton, field emission scanning electron microscope, energy dispersive X-ray spectroscope, and Fourier transform infrared spectroscope were employed to characterize the annealed powders after immersion. The powder with an amorphous structure induced hydroxyapatite formation on their surfaces after immersion for 15 days.     

Influence of SrO substitution for CaO on the properties of bioactive glass S53P4

Commercial melt-quenched bioactive glasses consist of the oxides of silicon, phosphorus, calcium and sodium. Doping of the glasses with oxides of some other elements is known to affect their capability to support hydroxyapatite formation and thus bone tissue healing but also to modify their high temperature processing parameters. In the present study, the influence of gradual substitution of SrO for CaO on the properties of the bioactive glass S53P4 was studied. Thermal analysis and hot stage microscopy were utilized to measure the thermal properties of the glasses. The in vitro bioactivity and solubility was measured by immersing the glasses in simulated body fluid for 6 h to 1 week. The formation of silica rich and hydroxyapatite layers was assessed from FTIR spectra analysis and SEM images of the glass surface. Increasing substitution of SrO for CaO decreased all characteristic temperatures and led to a slightly stronger glass network. The initial glass dissolution rate increased with SrO content. Hydroxyapatite layer was formed on all glasses but on the SrO containing glasses the layer was thinner and contained also strontium. The results suggest that substituting SrO for CaO in S53P4 glass retards the bioactivity. However, substitution greater than 10 mol% allow for precipitation of a strontium substituted hydroxyapatite layer.     

The processing and characterization of animal-derived bone to yield materials with biomedical applications. Part II: milled bone powders, reprecipitated hydroxyapatite and the potential uses of these materials

Further studies on the processing and use of animal-bone-derived calcium phosphate materials in biomedical applications are presented. Bone powders sourced either from the direct crushing and milling of bovine, ovine and cervine bone or after being subjected to defatting and acid digestion/NaOH reprecipitation and sodium hypochlorite hydrogen peroxide treatment of animal bones were characterized using Fourier transform infra-red (FTIR) spectroscopy, ¹³C solid state magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, atomic absorption (AA) and inductively coupled plasma (ICP) spectrometric techniques. Bone powders were trialled for their potential use as a substrate for phosphine coupling and enzyme immobilization as well as a feedstock powder for plasma spraying on titanium metal substrates. Results indicated that enzyme immobilization by phosphine coupling could be successfully achieved on milled cervine bone with the immobilized enzyme retaining some activity. It was found that the presence of impurities normally carried down with the processing of the bone materials (viz., fat and collagen) played an important role in influencing the adsorbency and reactivity of the powders. Plasma spraying studies using reprecipitated bovine-derived powders produced highly adherent coatings on titanium metal, the composition of which was mostly hydroxyapatite(Ca10(PO₄)₆(OH)₂) with low levels of -tricalcium phosphate (-Ca₃(PO₄)2) and tetracalcium phosphate (Ca₄P₂O₉) also detected. In general, animal derived calcium phosphate materials constitute a potentially cheaper source of calcium phosphate materials for biomedical applications and make use of a largely under-utilized resource from abattoir wastes. © 2000 Kluwer Academic Publishers     

Composite formation from hydroxyapatite with sodium and potassium salts of polyphosphazene

The low temperature synthesis of composites potentially suitable as bone substitutes which form in vivo, was investigated. The composites were comprised of stoichiometric hydroxyapatite (SHAp) and water-soluble poly phosphazenes. These constituents were selected because of their biocompatibility, and were mixed as powders with a phosphate buffer solution (PBS) to form the composites. The effects of poly[bis(sodium carboxylatophenoxy)phosphazene] (Na-PCPP) or poly[bis(potassium carboxylatophenoxy) phosphazene] (K-PCPP) on stoichiometric hydroxyapatite (SHAp) formation from tetracalcium phosphate and anhydrous dicalcium phosphate were assessed. The kinetics and reaction chemistries of composite formation were followed by isothermal calorimetry, X-ray diffraction, infrared spectroscopy and scanning electron microscopy. In the presence of 1% by weight of polyphosphazenes, composites comprised of SHAp and calcium cross-linked polymer salts were formed. Thus a mechanism for binding between polymer chains was established. Elevated proportions (5 and 10% by weight) of polyphosphazene, however, resulted in the inhibition of SHAp formation. This is attributed to the formation of viscous polymer solution coatings on the calcium phosphate precursors, retarding their reaction, and consequently inhibiting SHAp formation.     

Variation of crystal structure of hydroxyapatite in calcium phosphate cement by the substitution of strontium ions

New routes were used to introduce strontium into calcium phosphate cement in the present article. The study showed that by mixing 50 wt% amorphous calcium phosphate + amorphous strontium phosphate and 50 wt% dicalcium phosphate dihydrate, hydroxyapatite and Sr-hydroxyapatite precipitated separately in the hydrated cement; whereas, by mixing 50 wt% Sr- amorphous calcium phosphate and 50 wt% dicalcium phosphate dihydrate, strontium can be doped into hydroxyapatite lattice and increase the lattice dimensions and lattice volume. The strontium substituted calcium phosphate cement has potential for use in orthopedic surgeries.     

Evaluation of osteoinduction and proliferation on nano-Sr-HAP: a novel orthopedic biomaterial for bone tissue regeneration

Hydroxyapatite (HAP), a CaP compound similar to the mineral phase present in bone, has excellent biocompatibility but little osseous inductivity. In this study, we evaluated the novel nano-Sr-HAP, in which the calcium of hydroxyapatite was substituted with strontium, which acts as a bone-forming agent. Its biocompatibility and osteoinduction were assayed using marrow mesenchymal stem cells (MSCs) and osteoblasts (OBs) in vitro. We were able to demonstrate that nano-Sr-HAP supported increased OB cell adhesion, proliferation and viability up to 4 days in culture when compared with nano-HAP. MSCs cultured with nano-Sr-HAP showed higher alkaline phosphatase (ALP) activity. More extracellular mineralized nodules were found with nano-Sr-HAP compared to nano-HAP, especially in images of ALP staining. We suggest that nano-Sr-HAP powders possess osteoconductive and osteoinductive properties and have the potential to be used in the repair of bone defects caused by osteoporotic fractures.     

Influence of ripening time on the properties of hydroxyapatite-calcium carbonate powders

80 wt % hydroxyapatite + 20 wt % calcium carbonate composite powders have been synthesized through precipitation from aqueous solutions. Increasing the ripening time of the precipitate in the mother liquor leads to partial calcium carbonate dissolution and the formation of carbonate-substituted hydroxyapatite and improves its crystallinity. In addition, the specific surface area of the powders increases from 83.2 to 238.7 m²/g. The powders ripened for 14 and 21 days show the best sinterability.     

Influence of strontium for calcium substitution on the glass–ceramic network and biomimetic behavior in the ternary system SiO<Subscript>2</Subscript>–CaO–MgO

Strontium (Sr) enhances bone formation both in vitro and in vivo, while it reduces bone resorption. Thus, Sr incorporation in bioactive glass–ceramic scaffolds for bone tissue regeneration could further enhance osteogenesis. The aim of this work was the synthesis, characterization and investigation of the apatite-forming ability in inorganic environment of two sol–gel-derived bioactive Sr-containing glass–ceramic materials with 5 and 10% of SrO. The thermal properties of the synthesized materials were studied using differential thermal analysis (TG–DTA). The apatite-forming ability test was conducted in SBF for various immersion times for both thermally treated and untreated samples. The characterization of the samples before and after immersion in SBF was performed with Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD) and scanning electron microscopy with associated energy-dispersive spectroscopy. FTIR spectra revealed that all synthesized glass–ceramic materials presented the characteristic bands of silicate glasses, while XRD identified various crystalline phases, mostly calcium silicates. Strontium is present in the form of strontium silicate in both as-received and thermally treated specimens, and Sr-diopside in the thermally treated specimens. The apatite-forming ability of the glass–ceramic materials was confirmed by the formation of a hydroxyapatite layer after 3 and 5 days of immersion in SBF on the surface of the untreated and thermally treated samples, respectively. The apatite layer, also, became thicker as the immersion time increased.     

Surface characterization and biological properties of regular dentin,demineralized dentin,and deproteinized dentin

Bone autografts are often used for reconstruction of bone defects; however, due to the limitations of autografts, researchers have been in search of bone substitutes. Dentin is of particular interest for this purpose due to high similarity to bone. This in vitro study sought to assess the surface characteristics and biological properties of dentin samples prepared with different treatments. This study was conducted on regular (RD), demineralized (DemD), and deproteinized (DepD) dentin samples. X-ray diffraction and Fourier transform infrared spectroscopy were used for surface characterization. Samples were immersed in simulated body fluid, and their bioactivity was evaluated under a scanning electron microscope. The methyl thiazol tetrazolium assay, scanning electron microscope analysis and quantitative real-time polymerase chain reaction were performed, respectively to assess viability/proliferation, adhesion/morphology and osteoblast differentiation of cultured human dental pulp stem cells on dentin powders. Of the three dentin samples, DepD showed the highest and RD showed the lowest rate of formation and deposition of hydroxyapatite crystals. Although, the difference in superficial apatite was not significant among samples, functional groups on the surface, however, were more distinct on DepD. At four weeks, hydroxyapatite deposits were noted as needle-shaped accumulations on DemD sample and numerous hexagonal HA deposit masses were seen, covering the surface of DepD. The methyl thiazol tetrazolium, scanning electron microscope, and quantitative real-time polymerase chain reaction analyses during the 10-day cell culture on dentin powders showed the highest cell adhesion and viability and rapid differentiation in DepD. Based on the parameters evaluated in this in vitro study, DepD showed high rate of formation/deposition of hydroxyapatite crystals and adhesion/viability/osteogenic differentiation of human dental pulp stem cells, which may support its osteoinductive/osteoconductive potential for bone regeneration.     

Synthesis and release of trace elements from hollow and porous hydroxyapatite spheres

It is known that organic species regulate fabrication of hierarchical biological forms via solution methods. However, in this study, we observed that the presence of inorganic ions plays an important role in the formation and regulation of biological spherical hydroxyapatite formation. We present a mineralization method to prepare ion-doped hydroxyapatite spheres with a hierarchical structure that is free of organic surfactants and biological additives. Porous and hollow strontium-doped hydroxyapatite spheres were synthesized via controlling the concentration of strontium ions in a calcium and phosphate buffer solution. Similarly, fluoride and silicon-doped hydroxyapatite spheres were synthesized. While spherical particle formation was attainable at low and high temperature for Sr-doped hydroxyapatite, it was only possible at high temperature in the F/Si-doped system. The presence of inorganic ions not only plays an important role in the formation and regulation of biological spherical hydroxyapatite, but also could introduce pharmaceutical effects as a result of trace element release. Such ion release results showed a sustained release with pH responsive behavior, and significantly influenced the hydroxyapatite re-precipitation. These ion-doped hydroxyapatite spheres with hollow and porous structure could have promising applications as bone/tooth materials, drug delivery systems, and chromatography supports.     

Microstructure and chemistry affects apatite nucleation on calcium phosphate bone graft substitutes

The bioactivity of calcium phosphate bone grafts of varying chemistry and strut-porosity was compared by determining the rate of formation of hydroxycarbonate apatite crystals on the material surface after being soaked in simulated body fluid for up to 30 days. Three groups of silicate-substituted hydroxyapatite material were tested, with each group comprising a different quantity of strut-porosity (23, 32, and 46 % volume). A commercially available porous β-tricalcium phosphate bone graft substitute was tested for comparison. Results indicate that strut-porosity of a material affects the potential for formation of a precursor to bone-like apatite and further confirms previous findings that β-tricalcium phosphate is less bioactive than hydroxyapatite.     

Nanostructured hydroxyapatite powders produced by a flame-based technique

In this work we reported the production of hydroxyapatite (HA) powder, one of the most studied calcium phosphates in the bioceramics field, using a cost-effective apparatus, composed by three major components: the atomization device, the pilot and main flames and finally the powder collector system. Calcium acetate and ammonium phosphate, diluted in ethanol and water, were used as salts in the precursor solution. The Ca/P molar ratio in the precursor solution was 1.65, equivalent to biological hydroxyapatite. After its production and collection, HA powder was calcined at 600 °C for 2 h. X-ray diffraction analysis pointed to the formation of crystalline hydroxyapatite powders. Carbonate was identified in the powders by Fourier-transform infrared (FTIR) spectroscopy. Scanning electronic microscopy (SEM) showed that the powders were composed of spherical primary particles and secondary aggregates, with the morphology unchanged after calcination. By transmission electronic microscopy (TEM), it was observed that the crystallite size of the primary particles was 24.8 ± 5.8 nm, for the calcined powder. The specific surface area was 15.03 ± 6.4 and 26.50 ± 7.6 m²/g, for the as-synthetized and calcined powder respectively.     

Effect of fuel characteristics on synthesis of calcium hydroxyapatite by solution combustion route

The effect of fuel characteristics on the processing of nano sized calcium hydroxyapatite (HA) fine powders by the solution combustion technique is reported. Urea, glycine and glucose were used as fuels in this study. By using different combinations of urea and glycine fuels and occasional addition of small amounts of highly water-soluble glucose, the flame temperature (T _f) of the process as well as product characteristics could be controlled easily. The powders obtained by this modified solution combustion technique were characterized by XRD, FTIR spectroscopy, SEM, FESEM-EDX, particle size analyser (PSD) and specific surface area (SSA) measurements. The particle size of phase pure HA powder was found to be <20 nm in this investigation. The effects of glucose addition with stoichiometric (μ = 1) and fuel excess (μ > 1) urea and glycine precursor batches were investigated separately.     

Effect of Fuel on the Synthesis and Properties of Poly(methyl methacrylate) Modified SrFe12O19 Nanoparticles

Nanosized strontium hexaferrite (SrFe₁₂O₁₉) has been synthesized by citrate, urea, oxalic, and glycine precursor via a sol-gel route with poly(methyl methacrylate) (PMMA) as a templating agent. Crystal structure, morphology, and magnetic properties of as-synthesized nanoparticles were characterized by XRD, SEM, FT-IR, and VSM techniques. The formation of strontium hexaferrite and its crystallite size in presence of different fuels were compared. The influence of different fuels was reflected on the phase purity, morphology of the final powders as well as the magnetic properties. Magnetic measurements revealed that samples prepared by citric acid and glycine as fuel have high specific saturation magnetization and moderate coercivity, while urea and oxalic acid fuels resulted in low phase purity, and thus inferior magnetic properties.     

Biomimetic Mg-substituted hydroxyapatite: from synthesis to in vivo behaviour

The incorporation of magnesium ions (in the range 5–10 mol% in respect to Ca) into the hydroxyapatite structure, which is of great interest for the developing of artificial bone, was performed using magnesium chloride, calcium hydroxide and phosphoric acid, as reactants. Among the synthesized powders, the synthetic HA powder containing 5.7% Mg substituting for calcium was selected, due to its better chemico-physical features, and transformed into granules of 400–600 μm, for biocompatibility tests (genotoxicity, carcinogenicity, toxicity, in vitro cytotoxicity and in vivo skin irritation-sensitization tests). In vivo tests were carried out on New Zealand White rabbits using the granulate as filling for a femoral bone defect: osteoconductivity and resorption were found to be enhanced compared to commercial stoichiometric HA granulate, taken as control.     

Physico-chemical and in vitro biological evaluation of strontium/calcium silicophosphate glass

Strontium is known to reduce bone resorption and stimulate bone formation. Incorporation of strontium into calcium phosphate bioceramics has been widely reported. In this work, calcium and calcium/strontium silicophosphate glasses were synthesized from the sol–gel process and their rheological, thermal, and in vitro biological properties were studied and compared to each other. The results showed that the gel viscosity and thus the rate of gel formation increased by using strontium in glass composition and by increasing aging temperature. In strontium-containing glass, the crystallization temperature increased and the type of the crystallized phase was different to that of strontium-free glass. Both glasses favored precipitation of calcium phosphate layer when they were soaked in simulated body fluid; however strontium seemed to retard the rate of precipitation slightly. The in vitro biodegradation rate of the strontium/calcium silicophosphate glass was higher than that of strontium-free one. The cell culture experiments carried out using rat calvaria osteoblasts showed that the incorporation of strontium into the glass composition stimulated proliferation of the cells and enhanced their alkaline phosphatase activity, depending on cell culture period.