Simulated Body Fluid Treatment Research Articles

The Fabrication of Biomimetic Chitosan Scaffolds by Using SBF Treatment with Different Crosslinking Agents.

In this study, a chitosan substrate was modified by simulated body fluid (SBF) treatment, in which the effect of the chosen crosslinking agent was investigated. Two crosslinking agents, glutaraldehyde (GA) and sodium tripolyphosphate (TPP), were used before the SBF process. By using TPP as the crosslinking agent, the Ca/P ratio and the degree of crystallinity were very close to the natural bone matrix. On the contrary, the substrate properties were very different from natural bone when the crosslinking agent GA was used. The results indicate that the produced substrates were more biomimetic when the TPP was applied. On the SBF-modified chitosan substrates with TPP crosslinking, the cultured osteoblastic cells expressed better proliferation, mitochondria activity and differentiation ability. The chitosan crosslinked using TPP was a good template in the SBF process, which resulted in a highly biomimetic layer. This biomimetic substrate possesses excellent biocompatibility and osteoconduction ability, promising high potential in the promotion of bone tissue engineering.

Synthesis and characterization of hydroxyapatite/β-tricalcium phosphate nanocomposites using microwave irradiation

Microwave assisted synthesis method is a relatively new approach employed to decrease synthesis time and form a more homogenous structure in biphasic calcium phosphate bioceramics. In this study, nanocrystalline HA/β-TCP composites were prepared by microwave assisted synthesis method and, for comparison reason, by conventional wet chemical methods. The chemical and phase composition, morphology and particle size of powders were characterized by FTIR, XRD and SEM, respectively. The use of microwave irradiation resulted in improved crystallinity. The amount of hydroxyapatite phase in BCP ranged from 5% to 17%. The assessment of bioactivity was done by soaking of powder compacts in simulated body fluid (SBF). The decreasing pH of the solution in the presence of β-TCP indicated its biodegradable behavior. Rod-like hydroxyapatite particles were newly formed during the treatment in SBF for microwave assisted substrate synthesis. In contrast, globular particles precipitate under same conditions if BCP substrates were synthesized using conventional wet chemical methods.

SiO2−P2O5−CaO Glasses and Glass-Ceramics with and without ZnO: Relationships among Composition, Microstructure, and Bioactivity

Bioactive glasses and glass-ceramics of the system SiO2−P2O5−CaO with and without zinc were synthesized by sol−gel with the aim to address the influence of both the chemical composition and the microstructure on the growth of hydroxycarbonate apatite (HCA) following soaking in simulated body fluid (SBF). In the standard compositions 58S, 70S, and 80S the silica precursor was also partially replaced with a nanoscale silica powder (HiSil) in order to separate the contributions of porosity and composition. The combined use of XRPD, N2 absorption measurements, and SEM allowed us to establish quantitative correlations among the above-mentioned quantities. We found that the HCA formation rate increases with the silica content and that an important role is played by the specific surface area of the samples. Notably, the addition of even small quantities (0.4 wt %) of ZnO at the expenses of both CaO and P2O5 in the 58S glass leads to the increase, with respect to the 58S sample, of both the HCA formation rate and the HCA content after 8 days of treatment in SBF. A preliminary result revealed that sample with 0.4 wt % ZnO displayed good in vitro biocompatibility as shown by LDH assay.

In vitro biomineralization of glutaraldehyde crosslinked chitosan/glutamic acid films

In vitro biomineralization of glutaraldehyde crosslinked chitosan/glutamic acid films were studied. IR and ESCA (electron spectroscopy for chemical analysis) determinations confirm that chitosan and glutamic acid are successfully crosslinked by glutaraldehyde to form chitosan-glutamic acid surfaces. Composite films were soaked in saturated Ca(OH)2 solution for 8 d and then immersed in simulated body fluid (SBF) for more than 20 d. Morphological characterizations and structure of calcium phosphate coatings deposited on the films were studied by SEM, XRD, and EDAX (energy dispersive X-ray analysis). Initially, the treatment in SBF results in the formation of single-layer calcium phosphate particles over the film surface. As immersion time increases, further nucleation and growth produce the simulated calcium-carbonate hydroxyapatite coating. ICP results show Ca/P ratio of calcium phosphate coating is a function of SBF immersion time. The inducing of glutamic acid improves the biomineralization property of chitosan films.

Biomimetic nanofibrous gelatin/apatite composite scaffolds for bone tissue engineering

Mimicking certain features (e.g. nanoscale topography and biological cues) of natural extracellular matrix (ECM) is advantageous for the successful regeneration of damaged tissue. In this study, nanofibrous gelatin/apatite (NF-gelatin/apatite) composite scaffolds have been fabricated to mimic both the physical architecture and chemical composition of natural bone ECM. A thermally induced phase separation (TIPS) technique was developed to prepare nanofibrous gelatin (NF-gelatin) matrix. The NF-gelatin matrix mimicked natural collagen fibers and had an average fiber diameter of about 150 nm. By integrating the TIPS method with porogen leaching, three-dimensional NF-gelatin scaffolds with well-defined macropores were fabricated. In comparison to Gelfoam ® (a commercial gelatin foam) with similar pore size and porosity, the NF-gelatin scaffolds exhibited a much higher surface area and mechanical strength. The surface area and compressive modulus of NF-gelatin scaffolds were more than 700 times and 10 times higher than that of Gelfoam ®, respectively. The NF-gelatin scaffolds also showed excellent biocompatibility and mechanical stability. To further enhance pre-osteoblast cell differentiation as well as improving mechanical strength, bone-like apatite particles (<2 μm) were incorporated onto the surface of NF-gelatin scaffolds via a simulated body fluid (SBF) incubation process. The NF-gelatin/apatite scaffolds 5 days after SBF treatment showed significantly higher mechanical strength than NF-gelatin scaffolds 5 days after SBF treatment. Furthermore, the incorporated apatite in the NF-gelatin/apatite composite scaffold enhanced the osteogenic differentiation. The expression of BSP and OCN in the osteoblast–(NF-gelatin/apatite composite) constructs was about 5 times and 2 times higher than in the osteoblast–(NF-gelatin) constructs 4 weeks after cell culture. The biomimetic NF-gelatin/apatite scaffolds are, therefore, excellent for bone tissue engineering.

Cross-sectional analysis of the surface ceramic layer developed on Ti metal by NaOH-heat treatment and soaking in SBF

The surface structure developed on Ti metal after an NaOH and heat treatment and subsequent soaking in a simulated body fluid (SBF) was investigated using cross-sectional analysis involving SEM observations and EDX analysis, as well as an outer surface analysis involving SEM observations, thin-film X-ray diffraction, and Raman spectroscopy. A 1 μm-thick layer, which consisted of lathlike sodium hydrogen titanate (NaxH2-xTi3O7) elongated perpendicular to the surface, formed on the surface of the Ti metal after the initial NaOH treatment. This layer gradually changed into Ti metal at the boundary. The surface layer was densified by the subsequent heat treatment, accompanied by a transformation of the sodium hydrogen titanate into sodium titanate (Na2Ti6O13), rutile, and anatase. The scratch resistance of the surface layer significantly increased after the heat treatment. When this Ti metal with a modified surface was soaked in SBF, apatite began to precipitate in the interior of the surface layer, filled the interspaces of the lathlike phases to integrate with the latter giving a dense composite structure, and grew over the surface layer.

The &lt;i&gt;In Vivo &lt;/i&gt;Study of Biomimetic Chitosan Substrates’ Osteoinduction Effects

In this study, the chitosan scaffold was modified with the simulated body fluid (SBF) which would create a biomimetic layer on the interface between tissues and scaffolds for the bone formation. To investigate the in vivo osteoinduction, the chitosan scaffolds immersed in the SBF for different times were implanted into the calf muscle in male Wistar rats. The tissues blocks containing the scaffolds were harvested at different periods for bone induction assay and examined histologically. Hematoxyline and Eosin staining (H &amp; E staining), Masson’s Trichriome staining and alkaline phosphatase staining (ALPase), were performed for the observation of in vivo biocompatibility, collagen deposition and ALPase activity. Immunohistochemical staining of osteopontin (OPN) and bone sialoprotein (BSP) were used to examine gene expression of these bone proteins and to determine possible development stage of osteoinduction in the specimens with SBF modification. The results in this research suggested that the SBF modification would improve the biocompatibility of chitosan scaffolds, revealed by the decrease in foreign body reaction. With the SBF treatment, the expression of osteoblastic differentiation, including ALPase, OPN and BSP, would be also enhanced. Besides, the above tendencies would be more significant with the longer time for SBF immersion. In conclusion, the chitosan scaffolds modified by using SBF bioreactor would possess excellent biocompatibility and high potential in the promotion of bone regeneration.

An in vitro comparison of possibly bioactive titanium implant surfaces

The aim of the study was to compare Ca and P formation (CaP) and subsequent bone cell response of a blasted and four different possibly bioactive commercially pure (cp) titanium surfaces; 1. Fluoride etched (Fluoride), 2. Alkali-heat treated (AH), 3. Magnesium ion incorporated anodized (TiMgO), and 4. Nano HA coated and heat treated (nano HA) in vitro. Furthermore, to evaluate the significance of the SBF formed CaP coat on bone cell response. The surfaces were characterized by Optical Interferometry, Scanning Electron Microscopy (SEM) and X-ray Photoelectron Spectroscopy (XPS). CaP formation was evaluated after 12, 24 and 72 h in simulated body fluid (SBF). Primary human mandibular osteoblast-like cells were cultured on the various surfaces subjected to SBF for 72 h. Cellular attachment, differentiation (osteocalcin) and protein production (TGF-beta(1)) was evaluated after 3 h and 10 days respectively. Despite different morphological appearances, the roughness of the differently modified surfaces was similar. The possibly bioactive surfaces gave rise to an earlier CaP formation than the blasted surface, however, after 72 h the blasted surface demonstrated increased CaP formation compared to the possibly bioactive surfaces. Subsequent bone cell attachment was correlated to neither surface roughness nor the amount of formed CaP after SBF treatment. In contrast, osteocalcin and TGF-beta(1) production were largely correlated to the amount of CaP formed on the surfaces. However, bone response (cell attachment, osteocalcin and TGF-F production) on the blasted controls were similar or increased compared to the SBF treated fluoridated, AH and TiMgO surface.

Plasma processing for inducing bioactivity in stainless steel orthopaedic screws

The work presented in this paper is centred on applying plasma processing for inducing bioactivity (ability of a material to bond with bone) in otherwise bioinert stainless steel screws commonly used in orthopaedic surgery. As-received cortical stainless steel screws were hydroxylated using a patented two-step plasma process developed by the authors. The bioactivity of the screws thus processed was investigated in vitro by treating them with simulated body fluid (SBF). The SBF treatment resulted in a thin, continuous surface layer of hydroxyapatite (HA, calcium hydroxy phosphate) on the screws, thus confirming their bioactivity. The plasma processed screws were also compared with wet chemically processed screws for the growth of HA from SBF, and it was found that the HA layer on the plasma processed screws exhibited higher (complete) substrate coverage. The realisation of bioactive screws, in particular fine-threaded stainless steel screws, offers a major advancement towards the fixation of both internal and external orthopaedic implants.

Bioactive glass coatings affect the behavior of osteoblast-like cells

Functionally graded coatings (FGCs) of bioactive glass on titanium alloy (Ti6Al4V) were fabricated by the enameling technique. These innovative coatings may be an alternative to plasma-sprayed, hydroxyapatite-coated implants. Previously we determined that a preconditioning treatment in simulated body fluid (SBF) helped to stabilize FGCs [Foppiano S et al. Acta Biomater 2006;2(2):133–42]. The primary goal of this work was to assess the in vitro cytocompatibility of preconditioned FGCs with MC3T3-E1.4 mouse pre-osteoblastic cells. We evaluated cell adhesion, proliferation and mineralization on FGCs in comparison to uncoated Ti6Al4V and tissue culture polystyrene (TCPS). No difference in cell adhesion was identified, whereas proliferation was significantly different on all materials, being highest on FGCs followed by TCPS and Ti6Al4V. Qualitative and quantitative mineralization assays indicated that mineralization occurred on all materials. The amount of inorganic phosphate released by the mineralizing layers was significantly different, being highest on TCPS, followed by FGC and uncoated Ti6Al4V. The secondary objective of this work was to assess the ability of the FGCs to affect gene expression, indirectly, by means of their dissolution products, which was assessed by real-time reverse-transcription polymerase chain reaction. The FGC dissolution products induced a 2-fold increase in the expression of Runx-2, and a 20% decrease in the expression of collagen type 1 with respect to TCPS extract. These genes are regulators of osteoblast differentiation and mineralization, respectively. The findings of this study indicate that preconditioned FGCs are cytocompatible and suggest that future work may allow composition changes to induce preferred gene expression.

Hydrothermal–electrochemical CaTiO 3 coatings as precursor of a biomimetic calcium phosphate layer

The hydrothermal–electrochemical method was used to coat Ti and Ti6Al4V with calcium titanate CaTiO 3. The film exhibited a plate-like structure with several isolated pinholes around 100 nm in diameter. These coated samples were treated by a biomimetic procedure, immersing them in a simulated body fluid (SBF) solution for 5, 10 and 28 days. Characterization was performed by means of X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy and energy dispersive spectroscopy. The CaTiO 3-coated samples after the SBF treatment showed a significant increment in their calcium and phosphorous amounts as compared with SBF-treated samples with no previous CaTiO 3 coating; the latter exhibited only surface phosphate incorporation.

In vitro biomineralization of glutaraldehyde crosslinked chitosan films

The biomimetic approach was applied to study the in vitro biomineralization of series of the chitosan films crosslinked by glutaraldehyde. The deposited calcium phosphate coatings were studied using scanning electron microscopy and energy dispersive X-ray analysis. Initially, the treatment in simulated body fluid (SBF) results in the formation of single layer of calcium phosphate particles over the film surface. As immersion time in SBF increases, further nucleation and growth produce a simulated calcium phosphate coating. The Cal P molar ratio of the calcium phosphate increases with the immersion time, showing a rapid formation of calcium-deficient phosphate material from the phase of octac 1 alcium phosphate. The different glutaraldehyde crosslinking degree influences the morphology and magnitude of the calcium phosphate coatings on the surface of the chitosan films.

In vitro bioactivity and cytotoxicity of chemically treated glass fibers

Samples of a commercial glass fiber FM® (Fiber Max) were used to test the efficacy of a chemical sol-gel surface treatment to enhance their bioactivity. After treatment with tetraethoxysilane (TEOS), individual fiber samples were soaked into a simulated body fluid (SBF) solution, from which they were removed at intervals of 5 and 10 days. Micrographs obtained by scanning electron microscopy (SEM) analysis of samples chemically treated with TEOS revealed the formation of a hydroxyapatite (HA) coating layer after 5 days into SBF solution. Fourier transform infrared spectroscopic (FTIR) analyses confirmed that the coating layer has P-O vibration bands characteristic of HA. The in vitro cytotoxicity was evaluated using a direct contact test, minimum essential medium elution test (ISO 10993-5) and MTT assay. Fibers immersed in SBF and their extracts exhibited lower cytotoxicity than the controls not subjected to immersion, suggesting that SBF treatment improves the biocompatibility of the fiber.

Apatite-Forming Ability of Niobium Oxide Gels in a Simulated Body Fluid.

The essential prerequisite for an artificial material to show bone-bonding ability, i.e., bioactivity, is the formation of a bone-like apatite layer on its surface in a body environment. This can be reproduced in a simulated body fluid (SBF) with inorganic ion concentrations nearly equal to those of human blood plasma. It is known that some surface functional groups are effective for inducing heterogeneous apatite nucleation in SBF. Niobium is used clinically as a component in titanium alloys to improve their workability. In the present study, in order to investigate whether or not Nb-OH groups are effective for apatite nucleation, sol-gel-derived niobium oxide gels subjected to various heat treatments, and niobium metals subjected to various NaOH treatments were examined for their apatite-forming ability in SBF. The sol-gel-derived niobium oxide gels, both as-prepared, and heat-treated at 600°C, assumed an amorphous structure, and those heat-treated at 800 and 1000°C assumed orthorhombic and monoclinic structure, respectively. Apatite formation was observed on the niobium oxide gels both as-prepared, and heat-treated at temperatures below 800°C in SBF within one week, but not on the gel heat-treated at 1000°C. Niobium metals treated with the NaOH solutions did not form apatite on their surfaces, even after soaking in SBF for two weeks. It was confirmed that niobium metal formed a sodium niobate on its surface after the NaOH treatment, and that the sodium niobate formed a niobium oxide hydrogel by exchange of a Na+ ion with the H3O+ ion in SBF. Niobium oxide gels prepared by the sol-gel method have the ability to form an apatite layer on their surfaces in a body environment, when the gel has an amorphous phase structure, or an orthorhombic phase with abundant Nb-OH groups. Apatite deposition is not induced by Nb-OH groups formed on niobium metal after NaOH treatment and subsequent soaking in SBF. A niobium-based bioactive material can be developed through the design of a surface structure that effectively induces apatite nucleation.