Reinforcing ß-tricalcium phosphate scaffolds for potential applications in bone tissue engineering: impact of functionalized multi-walled carbon nanotubes.

Beta-tricalcium phosphate (ß-TCP) scaffolds manufactured through the foam replication method are widely employed in bone tissue regeneration. The mechanical strength of these scaffolds is a significant challenge, partly due to the rheological properties of the original suspension. Various strategies have been explored to enhance the mechanical properties. In this research, ß-TCP scaffolds containing varying concentrations (0.25-1.00 wt%) of multi-walled carbon nanotubes (MWCNT) were developed. The findings indicate that the addition of MWCNTs led to a concentration-dependent improvement in the viscosity of ß-TCP suspensions. All the prepared slurries exhibited viscoelastic behavior, with the storage modulus surpassing the loss modulus. The three time interval tests revealed that MWCNT-incorporated ß-TCP suspensions exhibited faster structural recovery compared to pure ß-TCP slurries. Introducing MWCNT modified compressive strength, and the optimal improvement was obtained using 0.75 wt% MWCNT. The in vitro degradation of ß-TCP was also reduced by incorporating MWCNT. While the inclusion of carbon nanotubes had a marginal negative impact on the viability and attachment of MC3T3-E1 cells, the number of viable cells remained above 70% of the control group. Additionally, the results demonstrated that the scaffold increased the expression level of osteocalcin, osteoponthin, and alkaline phosphatase genes of adiposed-derived stem cells; however, higher levels of gene expersion were obtained by using MWCNT. The suitability of MWCNT-modified ß-TCP suspensions for the foam replication method can be assessed by evaluating their rheological behavior, aiding in determining the critical additive concentration necessary for a successful coating process.

Design and Manufacture of Bone Cements Based on Calcium Sulfate Hemihydrate and Mg, Sr-Doped Bioactive Glass.

In the present study, a novel composite bone cement based on calcium sulfate hemihydrate (CSH) and Mg, Sr-containing bioactive glass (BG) as solid phase, and solution of chitosan as liquid phase were developed. The phase composition, morphology, setting time, injectability, viscosity, and cellular responses of the composites with various contents of BG (0, 10, 20, and 30 wt.%) were investigated. The pure calcium sulfate cement was set at approximately 180 min, whereas the setting time was drastically decreased to 6 min by replacing 30 wt.% glass powder for CSH in the cement solid phase. BG changed the microscopic morphology of the set cement and decreased the size and compaction of the precipitated gypsum phase. Replacing the CSH phase with BG increased injection force of the produced cement; however, all the cements were injected at a nearly constant force, lower than 20 N. The viscosity measurements in oscillatory mode determined the shear-thinning behavior of the pastes. Although the viscosity of the pastes increased with increasing BG content, it was influenced by the frequency extent. Pure calcium sulfate cement exhibited some transient cytotoxicity on human-derived bone mesenchymal stem cells and it was compensated by introducing BG phase. Moreover, BG improved the cell proliferation and mineralization of extracellular matrix as shown by calcein measurements. The results indicate the injectable composite cement comprising 70 wt.% CSH and 30 wt.% Mg, Sr-doped BG has better setting, mechanical and cellular behaviors and hence, is a potential candidate for bone repair, however more animal and human clinical evaluations are essential.

Physical, mechanical and in vitro biological evaluation of synthesized biosurfactant-modified silanated-gelatin/sodium alginate/45S5 bioglass bone tissue engineering scaffolds.

The aim of this study is to fabricate a highly porous scaffold based on gelatin/sodium alginate/45S5 bioglass with improved mechanical strength. Glycidoxypropyltrimethoxysilane (GPTMS) is used as a cross-linker and for the first time a nonionic bio-surfactant (Tween 80) is introduced to the composition to evaluate whether the essential properties of a suitable scaffold for bone substitution can be reached or not. The composite scaffolds are prepared through freeze-drying of suspension containing various ratios of gelatin/sodium alginate/45S5 bioglass. Characterization of fabricated scaffolds is carried out. SEM micrographs reveal that all samples are highly porous however incorporation of 1% v/v tween 80 results in well-shaped pores, with sizes ranging between 100 and 200 µm which is appropriate for tissue regeneration. Compressive strength of foamed scaffolds in contact with body solution has been enhanced from 0.37 to 1.41 MPa due to addition of tween 80. Foamed scaffold reinforced with tween 80 maintained its structural stability within 7 days immersion in simulated body fluid (SBF) despite the absorption of water 15 times its weight. Moreover, in vitro calcium phosphate precipitation is well observed on the surface of scaffolds.

Rheological evaluations and in vitro studies of injectable bioactive glass-polycaprolactone-sodium alginate composites.

Composite pastes composed of various amounts of melt-derived bioactive glass 52S4 (MG5) and polycaprolactone (PCL) microspheres in sodium alginate solution were prepared. Rheological properties in both rotatory and oscillatory modes were evaluated. Injectability was measured as injection force versus piston displacement. In vitro calcium phosphate precipitation was also studied in simulated body fluid (SBF) and tracked using scanning electron microscopy, X-ray diffraction and FTIR analyses. All composite pastes were thixotropic in nature and exhibited shear thinning behavior. The magnitude of thixotropy decreased by adding 10-30 wt% PCL, while further amounts of PCL increased it again. Moreover, the composites were viscoelastic materials in which the elastic modulus was higher than viscous term. The pastes which were just made of MG5 or PCL had poor injectability, whereas the composites containing both of these constituents exhibited reasonable injectability. All pastes revealed adequate structural stability in contact with SBF solution. In vitro calcium phosphate precipitation was well observed on the paste made of MG5 and somewhat on the pastes with 10-40 wt% PCL, however the precipitated layer was amorphous in nature. Overall, the produced composites may be appropriate as injectable biomaterials for non-invasive surgeries but more biological evaluations are essential.

Comparative study of mesenchymal stem cells osteogenic differentiation on low-temperature biomineralized nanocrystalline carbonated hydroxyapatite and sintered hydroxyapatite.

Hydroxyapatite with different characteristics in terms of morphology and chemistry were prepared via conventional sintering and low temperature biomimetic mineralization methods. The biomineralization route introduced nanocrystalline carbonate-substituted hydroxyapatite (n-CHA) with needle-like crystals ranging 20-30 nm whereas sintered HA (S-HA) comprised of polygonal grains ranging 2-5 µm. The response of fibroblastic cells was investigated using the extract of the samples whereas Wistar rat-derived mesenchymal stem cells (MSCs) were evaluated on top of each sample while maintaining in an osteogenic-free medium. The proliferation, activity, and morphology of adherent MSCs were determined at different culturing periods. The osteogenic differentiation of MSCs was also assayed by determining expression of runx2, osteonectin, osteopontin, and osteocalcin genes using real time-PCR analysis. The fibroblastic cells exhibited better proliferation rate at the presence of n-CHA compared to S-HA. Furthermore, the MSCs attached and spread well on both n-CHA and S-HA with better proliferation rate and alkaline phosphatase activity on n-CHA. Interestingly, the osteogenic differentiation of MSCs on n-CHA was confirmed by the expression of bone specific proteins whereas poor expression of these proteins was detected for the cells on S-HA. The results showed that the role of morphology, crystallinity, and chemistry of hydroxyapatite is crucial for osteogenesis differentiation of MSCs. The results predict osteoinductivity of n-CHA, because MSCs differentiation occurred at the absence of osteogenic medium. However, in vivo data are also required to support this suggestion.

Polymerizable nanoparticulate silica-reinforced calcium phosphate bone cement.

Bone cements based on calcium phosphate powder and different concentrations of colloidal silica suspensions were developed. Setting time and washout behavior of the cements were recorded and compared with those of a control group prepared by the same powder phase and distilled water as liquid. The phase composition, compressive strength, and morphology of the cements were determined after incubation and soaking in simulated body fluid. Proliferation of osteoblasts seeded on samples was also determined as a function of time. The results showed that the long setting time, poor compressive strength, and undesirable washout behavior of the cement made with distilled water were considerably improved by adding colloidal silica in a dose-dependent manner. On the basis of XRD and SEM results, both control group and nanosilica-added cements composed of nanosized apatite flakes after 7 days soaking, in addition to tetracalcium phosphate residual for the latter. It was found that the rate of hydraulic reactions that are responsible for conversion of the cement reactants to nanostructured apatite was increased by the presence of colloidal silica. Furthermore, the osteoblasts exhibited better proliferation on nanosilica added cements compared to control one. This study suggests better applied properties for nanosilica-added calcium phosphate cement compared to traditional cements.

Evaluation of colloidal silica suspension as efficient additive for improving physicochemical and in vitro biological properties of calcium sulfate-based nanocomposite bone cement.

In the present study new calcium sulfate-based nanocomposite bone cement with improved physicochemical and biological properties was developed. The powder component of the cement consists of 60 wt% α-calcium sulfate hemihydrate and 40 wt% biomimetically synthesized apatite, while the liquid component consists of an aqueous colloidal silica suspension (20 wt%). In this study, the above mentioned powder phase was mixed with distilled water to prepare a calcium sulfate/nanoapatite composite without any additive. Structural properties, setting time, compressive strength, in vitro bioactivity and cellular properties of the cements were investigated by appropriate techniques. From X-ray diffractometer analysis, except gypsum and apatite, no further phases were found in both silica-containing and silica-free cements. The results showed that both setting time and compressive strength of the calcium sulfate/nanoapatite cement improved by using colloidal silica suspension as cement liquid. Meanwhile, the condensed phase produced from the polymerization process of colloidal silica filled the micropores of the microstructure and covered rodlike gypsum crystals and thus controlled cement disintegration in simulated body fluid. Additionally, formation of apatite layer was favored on the surfaces of the new cement while no apatite precipitation was observed for the cement prepared by distilled water. In this study, it was also revealed that the number of viable osteosarcoma cells cultured with extracts of both cements were comparable, while silica-containing cement increased alkaline phosphatase activity of the cells. These results suggest that the developed cement may be a suitable bone filling material after well passing of the corresponding in vivo tests.