Concentrated Simulated Body Fluid Research Articles

Physico-chemical characterization of zirconia–titania composites coated with an apatite layer for dental implants

ObjectivesTo investigate the crystalline phases, morphological features and functional groups on the surface of sintered Y:TZP/TiO2 composite ceramics before and after the application of a biomimetic bone-like apatite layer. The effect of TiO2 content on the composite's characteristics was also evaluated. MethodsSamples of Y:TZP containing 0–30mol% TiO2 were synthesized by co-precipitation, followed by filtration, drying and calcination. The powders were uniaxially pressed and sintered at 1500°C/1h. To obtain biomimetic coatings the samples were exposed to sodium silicate solution and then to a concentrated simulated body fluid solution. The surfaces, before and after coating, were characterized by diffuse reflectance infrared Fourier transformed spectroscopy, X-ray diffraction analysis and scanning electron microscopy. ResultsThe surfaces of all Y:TZP/TiO2 samples were covered with a dense and uniform calcium phosphate layer with a globular microstructure. This layer was crystalline for specimens with 30% of TiO2 and amorphous for specimens with 0 and 10% of TiO2. Chemical analysis indicated that this layer was composed of type A carbonate apatite. Among the materials tested, the composite with 10% of TiO2 showed the best overall chemical and physical features, such as higher density and more cohesive amorphous apatite layer. SignificanceY-TZP-based materials obtained in the present investigation by means of the successful association of a calcium phosphate biomimetic layer with small amounts TiO2 should be further explored as an option for ceramic dental implants with improved bioactivity.

Effects of Nanosecond Laser Fabrication on Bioactivity of Pure Titanium

We are developing surface modification techniques for dental implants with the aim of reducing the time required to realize good adhesion between bone and implant surfaces. A nanosecond Nd:YVO4 laser was used to modify the surfaces of commercially pure titanium (CP Ti) disks and their bioactivities were then evaluated. The surfaces of the CP Ti disks were covered by lines after laser treatment. This treatment created complex microasperities of titania with rutile and anatase crystal structures. This results in the formation of hydroxyapatite on surfaces immersed in 1.5-times concentrated simulated body fluid for 7 days, whereas no hydroxyapatite was observed on conventionally polished surfaces that were immersed under the same conditions. This indicates that laser treatment improves the bioactivity of CP Ti, which is a critical property for osseointegrated implants.

Fabrication of mineralized electrospun PLGA and PLGA/gelatin nanofibers and their potential in bone tissue engineering

Surface mineralization is an effective method to produce calcium phosphate apatite coating on the surface of bone tissue scaffold which could create an osteophilic environment similar to the natural extracellular matrix for bone cells. In this study, we prepared mineralized poly(d,l-lactide-co-glycolide) (PLGA) and PLGA/gelatin electrospun nanofibers via depositing calcium phosphate apatite coating on the surface of these nanofibers to fabricate bone tissue engineering scaffolds by concentrated simulated body fluid method, supersaturated calcification solution method and alternate soaking method. The apatite products were characterized by the scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), and X-ray diffractometry (XRD) methods. A large amount of calcium phosphate apatite composed of dicalcium phosphate dihydrate (DCPD), hydroxyapatite (HA) and octacalcium phosphate (OCP) was deposited on the surface of resulting nanofibers in short times via three mineralizing methods. A larger amount of calcium phosphate was deposited on the surface of PLGA/gelatin nanofibers rather than PLGA nanofibers because gelatin acted as nucleation center for the formation of calcium phosphate. The cell culture experiments revealed that the difference of morphology and components of calcium phosphate apatite did not show much influence on the cell adhesion, proliferation and activity.

Rapid Deposition of Hydroxyapatite on Mg-Alloy by Biomineralization Method

Rapid deposition of hydroxyapatite on Mg-alloy in concentrated simulated body fluid (5×SBF) and modified simulated body fluid (m-SBF) was investigated. By biomineralization method, hydroxyapatite coating was deposited on Mg-alloy with pre-calcification treatment. Scanning electron microscope (SEM), energy disperse spectroscopy (EDS) and X-ray diffraction instrument (XRD) were applied to analyze the deposition product of biomineralization and the related mechanism. The results showed that pre-calcification treatment on Mg-alloy can lead to a quite rapid deposition of hydroxyapatite. Ionic concentrations in SBF solutions affected the structure of hydroxyapatite greatly. A homogeneous plate-like apatite coating was induced on Mg alloy sample in m-SBF solution which is promising for the future practice.

Characterization of electrophoretic chitosan coatings on stainless steel

Electrophoretic chitosan deposits on stainless steel AISI 316 L were produced and characterized. The coating quality (thickness, defectiveness, corrosion protection ability) was seen to depend on the electric field used for EPD. Corrosion studies in concentrated simulated body fluid (SBF5) demonstrated that the surface characteristics of AISI 316 L can be positively influenced by the chitosan coating.

In vitro corrosion and mineralization of novel Ti–Si–C alloy

The in vitro electrochemical behaviour of a new titanium based α-alloy (Ti–0.5 wt% Si–0.65 wt% C), fabricated via casting and rapid cooling route, was determined using linear, Tafel, potentiodynamic polarization tests and electrochemical impedance spectroscopy (EIS), complemented with ex situ SEM-EDS analysis to evaluate the corrosion mechanism. The experimental results revealed that silicon and carbon, in addition to titanium, resulted in the enhancement of mechanical properties. The polarization tests confirmed that Ti–Si–C alloy possessed excellent corrosion resistance (a low corrosion current density of 0.033 μA cm −2), comparable to cp Ti and better than Ti6Al4V in phosphate buffered saline (PBS). The mechanism of corrosion was identified as selective dissolution of titanium solid solution matrix. EIS studies indicated the formation of a stable, passive oxide film on the alloy. Further, in vitro bioactivity was evaluated using mineralization tests i.e. by immersing the pre-treated alloy in a concentrated simulated body fluid (10× SBF). Chemical and microstructural characterization of the mineral layer, formed during immersion, revealed the deposition of fine, porous micron-sized globules of a phase rich in calcium-phosphate (Ca-P). In summary, the bulk properties and excellent in vitro electrochemical and mineralization behaviour of the as-cast Ti–Si–C alloy reveal a high potential for its application as load bearing metallic implants.

Rapid Mineralization of Electrospun Scaffolds for Bone Tissue Engineering

We investigated different techniques to enhance calcium phosphate mineral precipitation onto electrospun poly(L-lactide) (PLLA) scaffolds when incubated in concentrated simulated body fluid (SBF), 10×SBF. The techniques included the use of vacuum, pre-treatment with 0.1 M NaOH and electrospinning gelatin/PLLA blends as means to increase overall mineral precipitation and distribution throughout the scaffolds. Mineral precipitation was evaluated using environmental scanning electron microscopy, energy dispersive spectroscopy mapping and the determination of the mineral weight percents. In addition we evaluated the effect of the techniques on mechanical properties, cellular attachment and cellular proliferation on scaffolds. Two treatments, pre-treatment with NaOH and incorporation of 10% gelatin into PLLA solution, both in combination with vacuum, resulted in significantly higher degrees of mineralization (16.55 and 15.14%, respectively) and better mineral distribution on surfaces and through the cross-sections after 2 h of exposure to 10×SBF. While both scaffold groups supported cell attachment and proliferation, 10% gelatin/PLLA scaffolds had significantly higher yield stress (1.73 vs 0.56 MPa) and elastic modulus (107 vs 44 MPa) than NaOH-pre-treated scaffolds.

Bone‐like apatite‐coated chitosan scaffolds: Characterization and osteoblastic activity

AbstractIn this study, porous scaffolds were prepared from chitosan (2% w/v in acetic acid and deacetylation degree: DD > 85%) by freeze‐drying method, and freshly lyophilized scaffolds were stabilized with ethanol solutions. Bone‐like apatite formation on chitosan scaffolds was achieved by immersing the scaffolds into a novel concentrated simulated body fluid (10× SBF‐like solution) for different periods, i.e., 6 and 24 h. Scanning electron microscope views showed that the 6‐h treatment in 10× SBF‐like solution led to the formation of calcium phosphate nucleation sites on chitosan scaffolds, whereas the apatite particles showed characteristic cauliflower‐like morphology at the end of 24‐h treatment. X‐ray diffraction results supported the fact that mineral phase was made of hydroxyapatite. Osteogenic activities of untreated and SBF‐treated chitosan scaffolds were examined by preosteoblastic MC3T3 cell culture studies. The mitochondrial activity test showed that apatite‐coated scaffolds stimulated cell proliferation compared with uncoated scaffolds. Alkaline phosphatase and osteocalcine levels indicated that the differentiation of the cells on all scaffolds increased significantly from 15th day of culture to the 21th day of culture, especially for the cells on 24‐h SBF‐treated scaffolds. The results of this study indicated that 10× SBF‐like solution‐treated chitosan scaffolds may be evaluated for bone tissue engineering. POLYM. COMPOS., 31:1418–1426, 2010. © 2009 Society of Plastics Engineers

Avaliação da biocompatibilidade do compósito aço/filme bioativo SiO2-CaO para aplicação biomédica

Foi obtido um filme bioativo sol-gel do sistema SiO2-CaO através da mistura de precursor de silício (TEOS - tetraetil ortosilicato), precursor de cálcio (nitrato de cálcio tetrahidratado) e álcool em meio ácido. Após a mistura, a solução serviu para revestir o aço inoxidável através do método de imersão empregando baixa velocidade de emersão. O compósito obtido foi então submetido a tratamento térmico para densificação do filme a 400 ºC durante 1 h. A biocompatibilidade do compósito foi avaliada através de dois métodos no sistema in vitro: a) solução concentrada similar ao fluido fisiológico - SFC 1,5 - e b) cultura de células. Imagens de microscopia eletrônica de varredura e espectroscopia de energia dispersiva comprovaram a precipitação de precursor da hidroxiapatita na superfície do filme bioativo após exposição à solução SFC durante 3 semanas. Imagens de microscopia eletrônica de varredura confirmaram aderência, crescimento e espalhamento celular na superfície do filme bioativo sol-gel após 24 h de cultivo celular, empregando células VERO (ATCC CCL-81), sugerindo que o compósito é um material potencialmente aplicável nas áreas de medicina e odontologia. Resultados obtidos com o ensaio de viabilidade celular através de MTT [brometo de 3-(4,5-dimetiltiazol-2-YL)-2,5-difeniltetrazolio] indicaram total ausência de toxicidade na interface filme sol-gel/células VERO.

Polyurethane foams electrophoretically coated with carbon nanotubes for tissue engineering scaffolds

Carbon nanotubes (CNTs) were deposited on the surfaces of polyurethane (PUR) foams by electrophoretic deposition (EPD). The parameters of EPD were optimized in order to obtain homogeneous CNT coatings on PUR foams and adequate infiltration of the three-dimensional (3D) porous network. The microstructure of the composites was investigated by high-resolution scanning electron microscopy (HRSEM), revealing that optimal quality of the coatings was achieved by an EPD voltage of 20 V. The thermal properties of the CNT-coated specimens, determined by thermogravimetric analysis (TGA), were correlated to the foam microstructure. In vitro tests in concentrated simulated body fluid (1.5 SBF) were performed to study the influence of the presence of CNTs on the bioactivity of PUR-based scaffolds, assessed by the formation of calcium phosphate (CaP) compounds, e.g. hydroxyapatite (HA), on the foam surfaces. It was observed that CNTs accelerate the precipitation of CaP, which is thought to be due to the presence of more nucleation centres for crystal nucleation and growth, as compared with uncoated foams. Polyurethane foams with CNT coating have the potential to be used as bioactive scaffolds in bone tissue engineering due to their high interconnected porosity, bioactivity and nanostructured surface topography.

Modified Cotton Fiber Surface for Apatite Growth and Cell Affinity

AbstractIn order to bring widely distributed polysaccharides materials into more medical applications, cotton fiber surfaces were modified into substrates for apatite deposition. Using a solid phase reaction, cotton fibers were conveniently carboxylated in large scale. The carboxylated cotton fibers were coated by apatite in a biomimetic way. Through soaking in a concentrated simulated body fluid (SBF × 5), nano-size apatite particles rapidly and finely grew on the fiber surfaces. The nucleation and growth of apatite was investigated with the aid of scanning electron microscopy (SEM). In comparison to pure cotton, the cotton coated with apatite showed improved cell affinity to osteoblast-like cells.

Hydroxyapatite coating of cellulose sponge does not improve its osteogenic potency in rat bone

Regenerated cellulose sponges were coated biomimetically with hydroxyapatite to increase their osteogenic properties. Induction of apatite precipitation was carried out with bioactive glass in simulated body fluid (SBF) for 24 h and the final coating was carried out in 1.5 × concentrated SBF for 14 days. Biomimetically mineralized and non-mineralized sponges were then implanted into standard size femoral cortical defects of rats, and the invasion of bone into the implant was followed up to one year. The apatite coating did not improve the osteoconductive property of cellulose in this rat cortical defect model. In fact, it generated a strong and highly cellular inflammatory reaction and less osteoid tissue. The biomimetic implants contained more immunodetectable TGFβ1 (a strong stimulator of fibroblast activity) than untreated implants, and also bound more TGFβ1 in vitro, which could, at least in part, explain the fibrotic invasion of biomimetically mineralized sponges.

Formation of apatite on poly(α‐hydroxy acid) in an accelerated biomimetic process

Bonelike apatite coating was formed on poly(L-lactic acid) films and poly(glycolic acid) scaffolds within 24 h through an accelerated biomimetic process. The ion concentrations in the simulated body fluid (SBF) were nearly 5 times of those in the human blood plasma. The apatite formed was characterized by using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The apatite formed in 5SBFs was similar in morphology and composition to that formed in the classical biomimetic process using SBF or 1.5SBF and similar to that of natural bone. This indicated that the biomimetic apatite-coating process could be accelerated by using concentrated simulated body fluid at 37 degrees C. Besides saving time, the accelerated biomimetic process is particularly significant to biodegradable polymers. Some polymers that degrade too fast to be coated with apatite by a classical biomimetic process (e.g., PGA) could be coated with bonelike apatite in an accelerated biomimetic process.

The effect of biomimetic apatite structure on osteoblast viability, proliferation, and gene expression

The conventional biomimetic apatite coating process can be accelerated by immersing substrates into concentrated simulated body fluid (5× SBF) at 37°C to form an initial coating of apatite precursor spheres, and transform the precursors into plate-like apatite structures. Depending on processing parameters, different apatite structures can be created over the same substrate. The purpose of this study is to investigate the effects of the different apatite microenvironment on cell spreading, viability, proliferation, and gene expression. MC3T3-E1 preosteoblasts were cultured on five surfaces: conventional apatite (CA), precursor apatite spheres (PreA), large plate-like apatites (LgA), small plate-like apatites (SmA), and tissue culture grade polystyrene (TCPS). PreA induced significantly higher cell death during the first two weeks. TCPS supported more uniform spreading (1 day) and higher proliferation (2 weeks) than CA, LgA, and SmA. Apatites restricted spreading and promoted the extension of cellular projections along the textured surfaces under confocal microscopy observation. By 3 weeks, LgA induced highest expression of mature osteogenic markers osteocalcin (OCN) and bone sialoprotein (BSP) in both regular and osteogenic culture media based on quantitative real-time RT-PCR. The results of this study suggest differential cell responses to subtle changes in apatite microenvironment.

The effect of pH on the structural evolution of accelerated biomimetic apatite

The classic biomimetic apatite coating process can be accelerated by first immersing substrates into concentrated simulated body fluid, 5× SBF (SBF1), at 37°C, to form an initial coating of precursor apatite spheres, and subsequently transferring to a second 5× SBF (SBF2) solution which is devoid of crystal growth inhibitors to promote phase transformation of SBF1-derived precursor apatite spheres into final crystalline apatite plates. Since SBF1 governs the formation kinetics and composition of the initial precursor spheres, we hypothesized that the pH of the SBF1 solution will also influence the final structure of the SBF2-derived crystalline apatite. To test this hypothesis, polystyrene substrates were immersed into SBF1 with different pH (5.8 or 6.5), and then immersed into the identical SBF2 (pH=6.0). The resultant apatites exhibited similar 2 θ XRD peaks; FTIR spectra in terms of hydroxyl, phosphate and carbonate groups; and Ca/P atomic ratio (1.42 for SBF1 5.8 apatite; 1.48 for SBF1 6.5 apatite). SEM, TEM and electron diffraction show that while SBF1 6.5 (pH 6.5) precursor spheres transform into larger, single crystals plates, SBF1 5.8 (pH 5.8) precursor spheres developed minute, polycrystalline plate-like structures over predominantly spherical precursor substrate.

Fast Formation of Biomimetic Ca-P Coatings on Ti6Al4V

AbstractThe aim of this study was to accelerate the formation of biomimetic Calcium-Phosphate (Ca-P) coatings on Ti6Al4V by using a 5 times more concentrated Simulated Body Fluid (SBFx5) than the regular SBF. The production of SBFx5 was possible by decreasing the pH of the solution to approximately 6 with CO2 gas. The release of this mildly acidic gas allowed the formation of a Ca-P film after 4h of immersion at pH=6.8. The structure of this coating was an amorphous carbonated Ca-P. In addition, our experiments showed that the presence of Mg2+ was absolutely necessary for the Ca-P coating formation on Ti6Al4V substrate. Mg2+ is a crystal growth inhibitor and favored the heterogeneous nucleation. Furthermore, depth profile X-Ray Photoelectron Spectroscopy showed that Ca-P nucleation on the passive Titanium oxide (TiO2) passive layer was initiated by Ca2+ and Mg2+. The attachment of this Ca-P coating resulted probably from chemical bonds such as P-O-Ti and Ca-O-Ti. Ca was more present at the coating/substrate interface than P. Thereby Ca-O-Ti seems to be favored.