Continuous multilayered composite hydrogel as osteochondral substitute.

Cartilage is a highly organized avascular soft tissue that assembles from nano-to macro-scale to produce a complex structural network. To mimic cartilage tissue, we developed a stable multilayered composite material, characterized by a tailored gradient of mechanical properties. The optimized procedure implies chemical crosslinking of each layer directly onto the previous one and ensures a drastic reduction of the material discontinuities and brittleness. The multilayered composite was characterized by infrared spectroscopy, differential scanning calorimetry, thermogravimetry, and scanning electron microscopy in order to compare its physico-chemical characteristics with those of cartilage tissue. The rheological behavior of the multilayered composite was similar to that of human cartilage. Finally its cytocompatibility toward chondrocytes and osteoblasts was evaluated.

Osteopenic bone cell response to strontium-substituted hydroxyapatite.

Ionic substitution is a powerful tool to improve the biological performance of calcium phosphate based materials. In this work, we investigated the response of primary cultures of rat osteoblasts derived from osteopenic (O-OB) bone to strontium substituted hydroxyapatite (SrHA), and to hydroxyapatite (HA) as reference material, compared to normal (N-OB) bone cells. Strontium (Sr) and calcium (Ca) cumulative releases in physiological solution are in agreement with the greater solubility of SrHA than HA, whereas the differences between the two materials are levelled off in DMEM, which significantly reduced ion release. O-OB cells grown on SrHA exhibited higher proliferation and increased values of the differentiation parameters. In particular, Sr substitution increased the levels of proliferation, alkaline phosphatase, and collagen type I, and down-regulated the production of interleukin-6 of O-OB cells, demonstrating a promising future of SrHA in the treatment of bone lesions and defects in the presence of osteoporotic bone.

Ionic substitutions in calcium phosphates synthesized at low temperature.

Ionic substitutions have been proposed as a tool to improve the biological performance of calcium phosphate based materials. This review provides an overview of the recent results achieved on ion-substituted calcium phosphates prepared at low temperature, i.e. by direct synthesis in aqueous medium or through hydrolysis of more soluble calcium phosphates. Particular attention is focused on several ions, including Si, Sr, Mg, Zn and Mn, which are attracting increasing interest for their possible biological role, and on the recent trends and developments in the applications of ion-substituted calcium phosphates in the biomedical field.

Chemico-physical characterization of gelatin films modified with oxidized alginate.

The possibility of using low concentrations of dialdehyde alginate (ADA) to crosslink and stabilize gelatin films was investigated. The films were prepared from gelatin solutions at different concentrations (5, 10 and 15wt.%) containing different amounts of oxidized alginate (0, 1 and 3wt.% with respect to the weight of gelatin). The extent of crosslinking increases as a function of ADA concentration, up to about 23%. The presence of oxidized alginate provokes a significant reduction in the degree of swelling and of gelatin release in phosphate-buffered saline solution, enhancing the effect of gelatin concentration. Furthermore, the values of the Young's modulus, E, and of the stress at break, sigma(b), increase with increasing ADA concentration. The observed small, but appreciable, increase in thermal stability found by differential scanning calorimetric investigation is supported by X-ray diffraction results.

Effect of strontium and gelatin on the reactivity of alpha-tricalcium phosphate.

The hydrolysis reaction of alpha-tricalcium phosphate (alpha-TCP) is of great interest because of its widespread use in the preparation of biomaterials for hard tissue repair. The aim of this study was to investigate how this reaction is influenced by the presence of a bioactive ion, Sr(2+), and of a biopolymer, gelatin, which were previously reported to affect the setting reaction of alpha-TCP-based cements. Hydrolysis experiments were carried out at different Sr(2+) concentrations (0, 5, 10, 20 at.%) in solutions at different gelatin concentrations (0, 0.1, 0.5, 1.0 wt.%). The results indicate that Sr(2+) delays the conversion of alpha-TCP into calcium-deficient hydroxyapatite (CDHA). The structural and morphological modifications of CDHA obtained from solutions at increasing Sr(2+) concentrations indicate that during hydrolysis strontium enters the structure of CDHA, where it partially substitutes for calcium. On the contrary, alpha-TCP hydrolysis rate increases on increasing gelatin concentration. Gelatin promotes conversion of alpha-TCP into octacalcium phosphate, and strongly interacts with the nucleating and growing crystals.

Effect of Mg(2+), Sr(2+), and Mn(2+) on the chemico-physical and in vitro biological properties of calcium phosphate biomimetic coatings.

We previously developed a calcium phosphate (CaP) calcifying solution that allows to deposit a uniform layer of nanocrystalline apatite on metallic implants in a few hours. In this work we modified the composition of the CaP solution by addition of Sr(2+), Mg(2+), and Mn(2+), in order to improve the biological performance of the implants. The results of the investigation performed on the coatings, as well as on the powders precipitated in the absence of the substrates, indicate that both Sr(2+) and Mg(2+) reduce the extent of precipitation, although they are quantitatively incorporated into the nanocrystalline apatitic phase. The inhibitory effect on deposition is much more evident for Mn(2+), which completely hinders the precipitation of apatite and yields just a small amount of amorphous phosphate relatively rich in manganese content. Human osteoblast-like MG-63 cells cultured on the different materials show that the Mg(2+) and Sr(2+) apatitic coatings promote proliferation and expression of collagen type I, with respect to bare Ti and to the thin layer of amorphous phosphate obtained in the presence of Mn(2+). However, the relatively high content of Mn(2+) in the phosphate has a remarkable beneficial effect on osteocalcin production, which is even greater than that observed for Sr(2+).

Interaction of Sr-doped hydroxyapatite nanocrystals with osteoclast and osteoblast-like cells.

This article reports the effect of strontium incorporation into hydroxyapatite nanocrystals on bone cells response. Hydroxyapatite nanocrystals were synthesized at strontium contents of 0, 1, 3, 7 atom %. Strontium incorporation for calcium is confirmed by the linear increase of the unit cell parameters of hydroxyapatite, in agreement with the different ionic radii of the two ions. Moreover, strontium substitution slightly affects hydroxyapatite structural order and the shape of the nanocrystals. Osteoblast-like MG63 cells cultured on the nanocrystals display good proliferation and increased values of the differentiation parameters. In particular, when cultured on samples with Sr concentration in the range 3-7 atom %, osteoblasts display increased values of ALP activity, collagen type I, and osteocalcin production. Moreover, the osteoclast number on all the Sr-doped samples is significantly smaller than on hydroxyapatite, and it decreases on increasing strontium content. The data indicate that strontium stimulates osteoblast activity and exerts its inhibitory effect on osteoclast proliferation even when incorporated into hydroxyapatite.

Strontium-substituted hydroxyapatite coatings synthesized by pulsed-laser deposition: in vitro osteoblast and osteoclast response.

The increasing interest in strontium incorporation into biomaterials for hard tissue repair is justified by the growing evidence of its beneficial effect on bone. We successfully synthesized hydroxyapatite (HA) thin films with different extents of strontium substitution for calcium (0, 1, 3 or 7 at.%) by pulsed-laser deposition. The coatings displayed a granular surface and a good degree of crystallinity, which slightly diminished as strontium content increased. Osteoblast-like MG63 cells and human osteoclasts were cultured on the thin films up to 21 days. MG63 cells grown on the strontium-doped HA coatings displayed normal morphology, good proliferation and increased values of the differentiation parameters, whereas the number of osteoclasts was negatively influenced by the presence of strontium. The positive effect of the ion on bone cells was particularly evident in the case of coatings deposited from HA at relatively high strontium contents (3-7%), where significantly increased values of alkaline phosphatase activity, osteocalcin, type I collagen and osteoprotegerin/TNF-related activation-induced cytokine receptor ratio, and considerably reduced values of osteoclast proliferation, were observed.

In vitro culture of mesenchymal cells onto nanocrystalline hydroxyapatite-coated Ti13Nb13Zr alloy.

In this study we coated a new biocompatible, nanostructured titanium alloy, Ti13Nb13Zr, with a thin layer of hydroxyapatite nanocrystals and we investigated the response of human bone-marrow-derived mesenchymal cells. The coating was realized using a slightly supersaturated CaP solution, which provokes a fast deposition of nanocrystalline hydroxyapatite. A thin layer of deposition is appreciable on the etched Ti13Nb13Zr substrates after just 1.5 h soaking in the CaP solution, and it reaches a thickness of 1-2 mum after 3 h soaking. The coating seems thinner than that deposited on Ti6Al4V, which was examined for comparison, likely because of the different roughness profiles of the two etched alloys, and it is constituted of elongated HA nanocrystals, with a mean length of about 100 nm. Mesenchymal stem cells were seeded onto coated and uncoated Ti alloys and cultured for up to 35 days. Cell morphology, proliferation and differentiation were evaluated. The cells display good adhesion and proliferation on the uncoated substrates, whereas the presence of hydroxyapatite coating slightly reduces cell proliferation and induces differentiation of MSCs towards a phenotypic osteoblastic lineage, in agreement with the increase of the expression of osteopontin, osteonectin and collagen type I, evaluated by means of rt-PCR. Type I collagen expression is higher in Ti13Nb13Zr MSC culture compared to Ti6Al4V, standing for a more efficient extracellular matrix deposition.

Nanocrystalline hydroxyapatite-polyaspartate composites.

The direct synthesis of hydroxyapatite-poly-L-aspartic acid (HA-PASP) nanocrystals has been carried out in presence of increasing amounts of PASP in solution up to 56 mmol/l. WAXS, TEM, TGA, IR and chemical analyses were used to characterize the structure, morphology and composition of the products. PASP is quantitatively incorporated into HA crystals, provoking a reduction of the coherent length of the crystalline domains. Furthermore, composite crystals display a greater length/width ratio with respect to the control HA crystals, and show a remarkable trend towards aggregation. The broadening of the X-ray diffraction reflections indicate a reduction of the coherent length along the long dimension 002 and the cross section 310 of the apatite crystals. The comparison between the morphological and structural data allows to suggest a specific interaction between PASP and HA structure.

Interaction of acidic poly-amino acids with octacalcium phosphate.

Octacalcium phosphate (OCP) hydrolysis into hydroxyapatite (HA) has been investigated in aqueous solutions at different concentrations of poly-L-aspartate (PASP) and poly-L-glutamate (PGLU). In the absence of the polyelectrolytes, the transformation of OCP into HA is complete in 48 h. Both poly-L-aspartate and poly-L-glutamate inhibit OCP hydrolysis. However, PGLU displays a greater inhibiting effect, as a result of the different extent of phase transformation obtained at the same polyelectrolyte concentration. The inhibition takes place through polyelectrolyte adsorption on the (100) face of OCP crystals, which prevents the splitting of OCP crystals along their c-axis and the transformation into the final very long, needle-like, apatitic crystals.

Alpha-tricalcium phosphate hydrolysis to octacalcium phosphate: effect of sodium polyacrylate.

Alpha-Tricalcium phosphate (alpha-TCP) hydrolysis into octacalcium phosphate (OCP) has been investigated in phosphoric acid solution at different concentrations of sodium polyacrylate (NaPA). The hydrolysis process has been followed by powder X-ray diffraction, infrared absorption and scanning electron microscopy analyses. In the absence of the polyelectrolyte, alpha-TCP undergoes a complete transformation into OCP in 24 h. The presence of polyacrylate in solution inhibits the hydrolysis so that a NaPA concentration of 0.5 microm is sufficient to lengthen the time required to complete the hydrolysis to 4 days. The variation of Ca2+ concentration in the soaking solution suggests that the transformation occurs through alpha-TCP dissolution followed by OCP precipitation. The delayed OCP nucleation and growth in the presence of polyacrylate implies a preferential adsorption of the polyelectrolyte on the growing OCP crystals, which induces an anisotropic reduction of the coherence lengths of the perfect crystalline domains.

Bonelike apatite growth on hydroxyapatite-gelatin sponges from simulated body fluid.

In vitro bioactivity of gelatin sponges and hydroxyapatite-enriched gelatin sponges was tested through evaluation of the variations in their composition and morphology after soaking in simulated body fluid (1.5) for periods up to 21 days at 37 degrees C. The presence of hydroxyapatite inside the sponges promotes the deposition of bonelike apatite crystals. The deposits are laid down as spherical aggregates, with mean diameters increasing from about 1-2 microm, after 4 days of soaking in simulated body fluid solution, up to about 3.5 microm in the samples soaked for 21 days. Simultaneously, the relative amount of inorganic phase increases up to about 56% wt, leading to a composite material with a composition quite close to that of bone tissue. The inorganic phase is a poor crystalline carbonated apatite similar to trabecular bone apatite.

Effect of sodium polyacrylate on the hydrolysis of octacalcium phosphate.

Octacalcium phosphate (OCP) hydrolysis into hydroxyapatite (HA) has been investigated in aqueous solutions at different concentrations of sodium polyacrylate (NaPA). In the absence of the polyelectrolyte, OCP undergoes a complete transformation into HA in 48 h. The hydrolysis is inhibited by the polymer, which is significantly adsorbed on the crystals, up to about 22 wt.%. A polymer concentration of 10(-2) mM is sufficient to cause a partial inhibition of OCP to HA transformation, which is completely hindered at higher concentrations. The small platelet-like crystals in the TEM images of partially converted OCP can display electron diffraction patterns characteristic either of OCP single crystals or of polycrystalline HA, whereas the much bigger plate-like crystals exhibit diffraction patterns characteristic of OCP single crystals. The polyelectrolyte adsorption on OCP crystals is accompanied by an increase of their mean length and by a significant reduction of the coherence length of the perfect crystalline domains along the c-axis direction. It is suggested that the carboxylate-rich polyelectrolyte is adsorbed on the hydrated layer of the OCP (100) face, thus inhibiting its in situ hydrolysis into HA.

Biomimetic growth of hydroxyapatite on gelatin films doped with sodium polyacrylate.

Gelatin films were used as biomimetic substrates for the nucleation of hydroxyapatite from simulated body fluid (SBF). Stretching and presence of sodium polyacrylate appear to be essential factors for the specific nucleation and growth of hydroxyapatite crystals inside the films. After soaking in 1.5SBF for periods longer than 4 days, all the films display a completely calcified surface. However, the spherical aggregates on the film surfaces do not give any X-ray diffraction effect and exhibit a low Ca/P molar ratio, typical of amorphous calcium phosphate. The ordered deposition of crystalline hydroxyapatite has been verified to take place only in stretched polyacrylate--gelatin films. The crystals grow as tablets about 2 microns thick among the gelatin layers, with their crystallographic c-axes preferentially oriented parallel to the direction of orientation of the collagen molecules, thus resembling the parallel orientation of apatitic crystals and collagen fibrils in calcified biological tissues.