Hrvoje Ivanković

PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: morphology, mechanical properties and bioactivity.

In the present study, poly(ε-caprolactone)-coated hydroxyapatite scaffold derived from cuttlefish bone was prepared. Hydrothermal transformation of aragonitic cuttlefish bone into hydroxyapatite (HAp) was performed at 200°C retaining the cuttlebone architecture. The HAp scaffold was coated with a poly(ε-caprolactone) (PCL) using vacuum impregnation technique. The compositional and morphological properties of HAp and PCL-coated HAp scaffolds were studied by means of X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and scanning electron microscopy (SEM) with energy dispersive X-ray (EDX) analysis. Bioactivity was tested by immersion in Hanks balanced salt solution (HBSS) and mechanical tests were performed at compression. The results showed that PCL-coated HAp (HAp/PCL) scaffold resulted in a material with improved mechanical properties that keep the original interconnected porous structure indispensable for tissue growth and vascularization. The compressive strength (0.88MPa) and the elastic modulus (15.5MPa) are within the lower range of properties reported for human trabecular bones. The in vitro mineralization of calcium phosphate (CP) that produces the bone-like apatite was observed on both the pure HAp scaffold and the HAp/PCL composite scaffold. The prepared bioactive scaffold with enhanced mechanical properties is a good candidate for bone tissue engineering applications.

Correlation of the precursor type with densification behavior and microstructure of sintered mullite ceramics

Abstract The effect of alumina component in diphasic mullite precursors containing alkoxy-derived silica on the crystallization and sintering behavior of compacts was studied. The phenomena observed were characterized using differential thermal analysis (DTA), powder X-ray diffraction (XRD), dilatometry and transmission and scanning electron microscopy (TEM, SEM). In order to change the characteristics of as-prepared gels, the alumina source was varied while keeping the silica source constant. Al(NO 3 ) 3 ·9H 2 O, γ-Al 2 O 3 and boehmite (γ-AlOOH) were used as the alumina source and TEOS as the silica source. Clear differences were found in the microstructure of sintered samples derived from the precursors with aluminum nitrate nonahydrate in comparison to the samples containing γ-Al 2 O 3 or boehmite (γ-AlOOH). The former exhibited elongated mullite grains embedded into the “equiaxial mullite matrix”. This morphology is due to the overlapping of mullite crystallization and viscous flow sintering temperatures. Transient alumina, either added as γ-Al 2 O 3 or formed in situ by decomposition of boehmite, shifts the mullite formation above the sintering temperature, and enables formation of equiaxial mullite. The smaller are the transient alumina particles, the smaller are mullite grains of sintered bodies.

Crystallization kinetics of mullite from single-phase gel determined by isothermal differential scanning calorimetry

Abstract Transformation kinetics of single-phase gel with mullite composition was studied by isothermal differential calorimetry (DSC) in temperature range from 937 to 959°C. Single exotherm was observed for annealing temperatures below 947°C, and two overlapped exothermic peaks were seen above this temperature. According to XRD analysis, mullite was the only phase crystallized either under non-isothermal or isothermal heat treatment. Johnson-Mehl-Avrami (JMA) equation for nucleation and growth could not describe mullite crystallization adequately, even below 947°C. Using bimodal JMA-type model, that proposes mullite crystallization in two steps, the fitting was remarkably good in the whole temperature range. Obtained kinetics data do not allow one to characterize the gel, either as typical single phase one (nucleation-controlled process with two rate constants and small apparent activation energies), or as hybrid gel (mullite formation via spinel and high apparent activation energies). The rate constants were an order of magnitude smaller than is proposed for single phase gel. The apparent activation energies, however (Ea1 = 1053 ± 51 kJ/mol, and Ea2 = 1028 ± 22 kJ/mmol, were in great discrepancy to those already cited for single phase gels, but they were in very good agreement with data evaluated for diphasic and hybrid gels. Mullite a-axis length and effective fraction of mullite that is formed in the first and second step of the process provided an insight in the mechanism of mullite crystallization. It is assumed that not the nucleation and crystallization limitations, rather the phase separation is the controlling process in mullite formation from single phase gel under applied experimental conditions.

Preparation and characterization of nano-hydroxyapatite within chitosan matrix

Nano-composites that show some features of natural bone both in composition and in microstructure have been prepared by in situ precipitation method. Apatite phase has been prepared from cost-effective precursors (calcite and urea phosphate) within chitosan (CS) matrix dissolved in aqueous acetic acid solution. The compositional and morphological properties of composites were studied by means of Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) thermogravimetric analysis (TGA) and transmission electron microscopy (TEM). Depending on the reaction conditions (temperature, reaction time, glucose addition and pH control) in addition to hydroxyapatite (HA) as a major phase, octacalcium hydrogen phosphate pentahydrate (OCP) and dicalcium phosphate anhydrate (DCPD) were formed as shown by XRD and FTIR. Crystallite lengths of precipitated HA estimated by Scherrers equation were between 20 and 30 nm. A fibrous morphology (~400 nm) of HA observed by TEM indicates that HA nucleates on chitosan chains.

Crystallization kinetics of mullite formation in diphasic gels containing different alumina components

Abstract The crystallization kinetics of mullite formation in diphasic gels containing TEOS-derived amorphous silica and alumina component in different crystalline form and particle size has been studied using quantitative X-ray diffraction (QXRD) with 3:2 mullite as a standard and transmission electron microscopy (TEM). As the source of alumina component, aluminum nitrate nonahydrate, commercial γ-Al2O3 and boehmite (γ-AlOOH) were used. The results were consistent with previous studies which indicated that mullite forms initially by nucleation and growth. During the second slow stage of transformation, mullite with approximate 2:1 composition subsequently converts further into 3:2. This process is the fastest in the precursor with the smallest particle size of alumina, and the slowest in the sample with boehmite-derived alumina. In very fine scale of morphology the conversion of 2:1 mullite started even in the first stage of transformation.

PCL-coated hydroxyapatite scaffold derived from cuttlefish bone : in vitro cell culture studies

In the present study, we examined the potential of using highly porous poly(ε-caprolactone) (PCL)-coated hydroxyapatite (HAp) scaffold derived from cuttlefish bone for bone tissue engineering applications. The cell culture studies were performed in vitro with preosteoblastic MC3T3-E1 cells in static culture conditions. Comparisons were made with uncoated HAp scaffold. The attachment and spreading of preosteoblasts on scaffolds were observed by Live/Dead staining Kit. The cells grown on the HAp/PCL composite scaffold exhibited greater spreading than cells grown on the HAp scaffold. DNA quantification and scanning electron microscopy (SEM) confirmed a good proliferation of cells on the scaffolds. DNA content on the HAp/PCL scaffold was significantly higher compared to porous HAp scaffolds. The amount of collagen synthesis was determined using a hydroxyproline assay. The osteoblastic differentiation of the cells was evaluated by determining alkaline phosphatase (ALP) activity and collagen type I secretion. Furthermore, cell spreading and cell proliferation within scaffolds were observed using a fluorescence microscope.

In Situ Hydroxyapatite Content Affects the Cell Differentiation on Porous Chitosan/Hydroxyapatite Scaffolds

Highly porous chitosan/hydroxyapatite composite structures with different weight ratios (100/0; 90/10; 80/20; 70/30; 60/40; 50/50; 40/60) have been prepared by precipitation method and freeze-gelation technique using calcite, urea phosphate and chitosan as starting materials. The composition of prepared composite scaffolds was characterized by X-ray diffraction analysis and Fourier transformed infrared spectroscopy, while morphology of scaffolds was imaged by scanning electron microscopy. Mercury intrusion porosimetry measurements of prepared scaffolds have shown different porosity and microstructure regarding to the HA content, along with SEM observations of scaffolds after being immersed in physiological medium. The results of swelling capacity and compressive strength measured in Dulbecco’s phosphate buffer saline (DPBS) have shown higher values for composite scaffolds with lower in situ HA content. Viability, proliferation and differentiation of MC3T3-E1 cells seeded on different scaffolds have been evaluated by live dead assay and confocal scan microscopy. Our results suggest that the increase of HA content enhance osteoblast differentiation confirming osteogenic properties of highly porous CS/HA scaffolds for tissue engineering applications in bone repair.

Highly Porous Hydroxyapatite Ceramics for Engineering Applicatios

Highly porous hydroxyapatite (Ca10(PO4)6(OH)2, HA) was prepared through hydrothermal (HT) transformation of aragonitic cuttlefish bones (Seppia Officinalis L. Adriatic Sea) in the temperature range from 140°C to 220°C for 20 minutes to 48 hours. Mechanism of hydrothermal transformation of bones was investigated by DTA/TG analyzer coupled online with FTIR spectrometric gas cell equipment (DTA-TG-EGA-FTIR analysis), X-ray diffraction analysis (XRD) and scanning electron microscopy (SEM). DTA-TG-EGA-FTIR analysis have shown the release of CO2 at about 400°C, 680°C and 990°C. The first release could be attributed to organics not completely removed from the heat treated bones, and the second release to decomposition of unconverted aragonite, whereas, the third one could be attributed to CO3 2– groups incorporated in the structure of HA. The interconnecting porous morphology of the starting material (aragonite) was maintained during the HT treatment. The formation of dandelion-like HA spheres with diameter from 3 to 8 μm were observed, which further transformed into nanoplates and nanorods with an average diameter of about 200-300 nm and an average length of about 8-10 μm.

Crystallization of high-quartz solid solution in gahnite glass-ceramics

Abstract The crystallization of ZnO–(MgO)–Al2O3–B2O3–SiO2 glasses, having molar ratio ZnO/Al2O3 = 1 and variable content of MgO, with TiO2 or ZrO2 or both as nucleating agents, has been studied. Phase composition and microstructure of the obtained glass-ceramics were examined by XRD, SEM, EDX and TEM analyses. The main crystalline phase obtained is gahnite, ZnAl2O4. High-quartz solid solution, as an additional phase, crystallizes in the glasses containing ZrO2 or ZrO2 + TiO2 as nucleating agents. When ZrO2 is present alone, the unit cell parameter, a, of high-quartz solid solution decreases linearly with temperature. However, if TiO2 is added as another nucleating agent, a non-linear dependence of the parameter, a, on temperature occurs. Inside the primary separated droplet-shaped phase, from which gahnite precipitates, B2O3 is concentrated in the centre and extends radially to the phase boundary, inducing a specific microstructure of gahnite.

Preparation of Highly Porous Hydroxyapatite Ceramics from Cuttlefish Bone

Scaffold of hydroxyapatite for further tissue-engineering application was produced by hydrothermal treatment of cuttlefish bone originated aragonite at 200°C. Aragonite (CaCO3) monoliths were completely transformed into hydroxyapatite after 48 hours of HT treatment. The substitution of CO3- groups predominantly into the PO4 3- sites of the Ca10(PO4)6(OH)2 structure was suggested by FTIR spectroscopy. SEM micrographs have shown that the interconnected hollow structure with pillars connecting parallel lamellae in cuttlefish bone is maintained after conversion. Specific surface area (SBET) and total pore volume increased and mean pore size decreased by HT treatment.