Hydroxyapatite formation from cuttlefish bones: kinetics.

Highly porous hydroxyapatite (Ca(10)(PO(4))(6)·(OH)(2), HA) was prepared through hydrothermal transformation of aragonitic cuttlefish bones (Sepia officinalis L. Adriatic Sea) in the temperature range from 140 to 220°C for 20 min to 48 h. The phase composition of converted hydroxyapatite was examined by quantitative X-ray diffraction (XRD) using Rietveld structure refinement and Fourier transform infrared spectroscopy (FTIR). Johnson-Mehl-Avrami (JMA) approach was used to follow the kinetics and mechanism of transformation. Diffusion controlled one dimensional growth of HA, predominantly along the a-axis, could be defined. FTIR spectroscopy determined B-type substitutions of CO(3) (2-) groups. The morphology and microstructure of converted HA was examined by scanning electron microscopy. The general architecture of cuttlefish bones was preserved after hydrothermal treatment and the cuttlefish bones retained its form with the same channel size (~80 × 300 µm). The formation of dandelion-like HA spheres with diameter from 3 to 8 µm were observed on the surface of lamellae, which further transformed into various radially oriented nanoplates and nanorods with an average diameter of about 200-300 nm and an average length of about 8-10 µm.

Fibrin-chitosan composite substrate for in vitro culture of chondrocytes.

The aim of this study was to develop a biocompatible monolayer substrate based on fibrin and chitosan for in vitro culture of chondrocytes. Fibrin-chitosan composite substrates combined the proved cell adhesion properties of fibrin with the hydrophilicity and poor adhesion capacity of chitosan. Chitosan microspheres were produced by coacervation method, agglomerated within a fibrin network and subsequently crosslinked with genipin. The composite substrate was stable for 28 days of culture due to the high crosslinking density. Human chondrocytes cultured on the composite substrate were viable during the culture period. At the end of culture time (28 days) the composite substrate showed low cellular proliferation, 41% more collagen type II and 13% more production of sulfated glycosaminoglycans with respect to the amounts found at 14 days. The study revealed that dedifferentiated chondrocytes cultured in monolayer on the composite substrate can re-acquire characteristics of differentiated cells without using three-dimensional substrates or chondrogenic media.

In vitro 3D culture of human chondrocytes using modified ε-caprolactone scaffolds with varying hydrophilicity and porosity.

Two series of 3D scaffolds based on ε-caprolactone were synthesized. The pore size and architecture (spherical interconnected pores) was the same in all the scaffolds. In one of the series of scaffolds, made of pure ε-polycaprolactone, the volume fraction of pores varied between 60% and 85% with the main consequence of varying the interconnectivity between pores since the pore size was kept constant. The other scaffolds were prepared with copolymers made of a ε-caprolactone-based hydrophobous monomer and hydroxyethyl acrylate, as the hydrophilic component. Thus, the hydrophilicity and, presumably, the adhesion properties varied monotonously in the copolymer series while porosity was kept constant. A suspension of human chondrocytes in culture medium was injected in the 3D scaffolds and cultured in static conditions up to 28 days. SEM and immunofluorescence assays allowed characterizing cells and extracellular matrix inside the scaffolds after different culture times. To do that, cross sections of the scaffolds were observed by SEM and confocal microscopy. The quantity of cells inside the scaffolds decreases with a decrease of the volume fraction of pores, due to the lack of interconnectivity between the cavities. The scaffolds up to a 30% of hydrophilicity behave in a similar way than the hydrophobous; a further increase of the hydrophilicity rapidly decreases cell viability. In all the experiments production of collagen type I, type II, and aggrecan was found, and some cells were Ki-67 positive, showing that some cells are adhered to the pore walls and maintain their dedifferentiated phenotype even when cultured in three-dimensional conditions.

Surface modification of P(EMA-co-HEA)/SiO2 nanohybrids for faster hydroxyapatite deposition in simulated body fluid?

P(EMA-co-HEA)/SiO(2) nanocomposites with 0, 15 and 30 wt% of silica were obtained by copolymerization of ethyl methacrylate, EMA, and hydroxyethyl acrylate, HEA, during the simultaneous acid-catalyzed sol-gel polymerization of tetraethoxysilane, TEOS. A surface modification treatment was applied in order to reduce the induction time for hydroxyapatite (HAp) nucleation, combining a previous NaOH attack to increase the number of surface nucleating sites, and an alternate soaking process in Ca and P solutions to form apatite precursors, prior to the immersion in a simulated body fluid (SBF). The NaOH treatment was not effective by itself in shortening the HAp induction time. It introduced sodium carboxylates in the copolymer but hydrolyzed the silica network excessively, thus reducing the surface nucleating potential of its boundary silanols. Therefore, bioactivity was only due to the surface carboxylate groups of the organic phase. Maybe a controlled dissolution extent of the silica network so as to improve bioactivity could be attained by reducing the duration of the NaOH-treatment. This would be interesting in the hybrid with 30wt% of silica, because its dense silica network is not able to hydrolyze in SBF without any previous treatment, whereas the silica network in the hybrid with 15wt% of silica hydrolyzes at the surface promoting the deposition of HAp. The CaP treatment was able to coat the surfaces of the samples with a calcium phosphate layer within minutes. This amorphous calcium phosphate acted as HAp precursor, skipping the induction period in SBF.

Bioactive scaffolds mimicking natural dentin structure.

Organic scaffolds of poly(ethyl methacrylate-co-hydroxyethyl acrylate) [P(EMA-co-HEA)] 70/30 wt % ratio, with varying proportions of silica SiO(2) from 0 to 20 wt % and aligned tubular pores, were prepared using a fiber-templating fabrication method, with the aim of mimicking structure and properties of the mineralized tissue of natural dentin. Precursors of the copolymer and silica were simultaneously polymerized in a sol-gel process within the fiber template, which was eventually eliminated to generate homogeneously distributed parallel micrometer-sized pores in the material. Scaffolds of PEMA and PHEA were obtained by the same approach. The scaffolds were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and thermogravimetric analysis. The specific volume was determined by Archimedes' method and the porosity calculated from the geometry. The mechanical properties were analyzed in tensile and compressive modes. The bioactivity of the scaffolds with 15 wt % SiO(2) was tested by immersion in simulated body fluid (SBF) for 7 days followed by immersion in 2x SBF for 7 days. These scaffolds were afterwards characterized by SEM, energy dispersive spectroscopy, and compression assays. Percentages of silica above 10 wt % reinforced mechanically the copolymer, evidenced by the hindrance of the long range motions of the organic chains, altered shrinkage and swelling, and meanwhile conferred bioactivity to its surface. These tubular porous structures, which resemble natural dentin with regard to its structure and properties and induce the precipitation of apatite on their surfaces in vitro, are expected to facilitate the integration in the host mineralized tissue, to stimulate cell growth and to be useful as guiding scaffolds for in vivo dentin regeneration.

Acrylic scaffolds with interconnected spherical pores and controlled hydrophilicity for tissue engineering.

Polymer scaffolds are obtained in which the geometric characteristics (pore size, connectivity, porosity) and the physico-chemical properties of the resulting material can be controlled in an independent way. The interconnected porous structure was obtained using a template of sintered PMMA microspheres of controlled size. Copolymerization of hydrophobic ethyl acrylate and hydrophilic hydroxyethyl methacrylate comonomers took place in the free space of the template, different comonomer ratio gave rise to different hydrophilicity degrees of the material keeping the same pore architecture. The morphology of the resulting scaffolds was investigated by scanning electron microscopy (SEM), the porosity of the material calculated, and the mechanical properties compared with those of the bulk (non porous) material of the same composition.