Fiber reinforcement of a biomimetic bone cement.

In this study we investigated the influence of electrospun polymer fibers on the properties of a α-tricalcium phosphate/gelatin biomimetic cement. To this aim, we added different amounts of poly(L-lactic acid) and poly(lactide-co-glycolide) fibers to the cement composition. Fibers enrichment provoked a significant reduction of both initial and final setting times. Moreover electrospun polymer fibers slowed down the conversion of α-tricalcium phosphate into calcium deficient hydroxyapatite. As a result, the final cements were more compact than the control cement, because of the smaller crystal dimensions and reduced crystallinity of the apatitic phase. The compressive strength, σ(b), and Young's modulus, E, of the control cement decreased significantly after 40 days soaking in physiological solution, whereas the more compact microstructure enabled fiber reinforced cements to maintain their mechanical properties in the long term.

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.

Human osteoblast response to pulsed laser deposited calcium phosphate coatings.

Octacalcium phosphate (OCP) and Mn(2+)-doped carbonate hydroxyapatite (Mn-CHA) thin films were deposited on pure, highly polished and chemically etched Ti substrates with pulsed laser deposition. The coatings exhibit different composition, crystallinity and morphology that might affect their osteoconductivity. Human osteoblasts were cultured on the surfaces of OCP and Mn-CHA thin films, and the cell attachment, proliferation and differentiation were evaluated up to 21 days. The cells showed a normal morphology and a very good rate of proliferation and viability in every experimental time. Alkaline phosphatase activity was always higher than the control and Ti groups. From days 7 to 21 collagen type I production was higher in comparison with control and Ti groups. The level of transforming growth factor beta 1 (TGF-beta1) was lower at 3 and 7 days, but reached the highest values during following experimental times (14 and 21 days). Our data demonstrate that both calcium phosphate coatings favour osteoblasts proliferation, activation of their metabolism and differentiation.

Effect of added gelatin on the properties of calcium phosphate cement.

This study investigates the effect of gelatin on the setting time, compressive strength, phase evolution and microstructure of calcium phosphate cement. The composite cement powder (about 18 wt% gelatin, and 82 wt% alpha-tricalcium phosphate) was prepared from the solid compound obtained by casting a gelatin aqueous solution containing alpha-tricalcium phosphate. 5 wt% of CaHPO(4) x 2H(2)O were added to the powder before mixing with the liquid phase. Two cement formulations were prepared using two different liquid/powder ratios, and their properties compared with those of control samples, prepared without gelatin. The final setting time increases from 10 min to more than 45 min when the L/P ratio increases from 0.3 to 0.4 ml/g. The presence of gelatin accelerates the setting reaction, and improves the mechanical properties of the cements. The compressive strength increases with the setting reaction up to 10.7-14.0 MPa for the gelatin cements, whereas the control samples exhibit much lower values. The improved mechanical properties of the composite cements with respect to the controls can be related to their reduced total porosity and more compact microstructure.

Drawn gelatin films with improved mechanical properties.

Chain anisotropic distribution in gelatin films has been obtained by uniaxial stretching at constant relative humidity, followed by air drying and successive cross-linking with glutaraldehyde. The drawn samples have been characterized by mechanical tests, differential scanning calorimetry and scanning electron microscopy. The Young's modulus, E, and the stress at break, sigma(b), increase linearly with the draw ratio and reach values which are about five times those characteristic of undrawn samples. Furthermore, on stretching the alignment of the gelatin strands along the direction of deformation increases while the thickness of the layers decreases significantly. The renaturation level, that is the fraction of gelatin in a collagen-like structure, has been calculated as the ratio between the melting enthalpy of gelatin samples and that of tendon collagen. The results indicate that the improvement of mechanical properties achieved by drawn gelatin is closely related to the renaturation level. The experimental approach utilized to induce segmental orientation in gelatin films, allows to obtain anisotropic materials with improved mechanical properties in the direction of deformation, and can be usefully applied in the preparation of biomaterials.

Biocompatible nanocrystalline octacalcium phosphate thin films obtained by pulsed laser deposition.

We extended for the first time pulsed laser ablation to the deposition of octacalcium phosphate Ca8H2(PO4)6.5H2O (OCP) thin films. The depositions were performed with a pulsed UV laser source (lambda=248 nm, tau> or =20 ns) in a flux of hot water vapors. The targets were sintered from crystalline OCP powder and the laser ablation fluence was set at values of 1.5-2 J/cm2. During depositions the collectors, Si or Ti substrates, were maintained at a constant temperature within the range 20-200 degrees C. The resulting structures were submitted to heat treatment in hot water vapors for up to 6 h. The best results were obtained at a substrate temperature of 150 degrees C during both deposition and post-deposition treatment. High-resolution electron microscopy and XRD at grazing incidence indicated that the coatings obtained were made of nanocrystalline OCP. Cross-section TEM investigations showed that the coatings contained droplets stacked on Ti substrates as well as distributed across the entire thickness of the arborescence-like structure layers. The results of WST-1 assay, cell adherence, DNA replication, and caspase-1 activity confirmed the good biocompatibility of the coatings.

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.

A biomimetic gelatin-calcium phosphate bone cement.

The interest in new bone substitutes is rapidly increasing in the field of orthopedic surgery. A variety of calcium phosphate bone cement has been developed and different additives have been used to improve their biocompatibility and bioactivity. Following a biomimetic strategy aimed at reproducing bone characteristics, this study investigates the biological properties of a new gelatin enriched calcium phosphate cement (GEL-CP) that exhibits improved mechanical properties with respect to cement prepared without gelatin (C-CP). Human osteoblast MG63 were cultured on the surfaces of GEL-CP and were compared to cells cultured on C-CP samples, and on polystyrene of plate culture as control (C). Cell attachment, proliferation and differentiation were evaluated up to 21 days. SEM revealed that osteoblasts grown on GEL-CP showed a normal morphology and biological tests demonstrated very good rate of proliferation and viability in every experimental time. The presence of gelatin stimulated alkaline phosphatase activity, collagen and transforming growth factor 31 production. The data indicate that the new cement GEL-CP favors osteoblast proliferation, activation of their metabolism and differentiation. The remarkable improvement of the setting properties of the calcium phosphate cement due to the presence of gelatin suggest that the biomimetic composite material could be successfully applied as bone substitute.

Functionalization of biomimetic calcium phosphate bone cements with alendronate.

Bisphosphonates (BPs) are widely employed for the treatment of a variety of bone disorders. We have previously successfully added small amounts of BPs into calcium phosphate bone cements in order to enhance their bio-functionality. In this work we were able to increase greatly the amount of BP introduced in the cement, thanks to suitable modifications of composition. In particular, we utilized biomimetic alpha-tricalcium phosphate (alpha-TCP) cements at different gelatin contents (10, 15 and 20 wt.%) to introduce Disodium Alendronate up to a concentration of 25 mM. Due to the small liquid/powder ratio (0.22 ml/g) the lengthening of the setting times due to alendronate is quite modest. The rate of transformation of alpha-TCP into calcium deficient hydroxyapatite slightly decreases as a function of alendronate content, whereas it increases with increasing gelatin concentration. Moreover, relatively high alendronate concentrations provoke significant reduction of the compressive strength of the cements. The results of in vitro tests indicate that alendronate-containing cements significantly affect osteoclast proliferation and differentiation, whereas they promote osteoblast differentiation, to an extent which depends on cement composition.

Alendronate and Pamidronate calcium phosphate bone cements: setting properties and in vitro response of osteoblast and osteoclast cells.

We have investigated the effect of Alendronate and Pamidronate, two bisphosphonates widely employed for the treatment of pathologies related to bone loss, on the setting properties and in vitro bioactivity of a calcium phosphate bone cement. The cement composition includes alpha-tricalcium phosphate (alpha-TCP) (90 wt%), nanocrystalline hydroxyapatite (5 wt%) and CaHPO(4) x 2H(2)O (5 wt%). Disodium Alendronate and disodium Pamidronate were added to the liquid phase (bidistilled water) at two different concentrations: 0.4 and 1mM (AL0.4, AL1.0, PAM0.4, PAM1.0). Both the initial and the final setting times of the bisphosphonate-containing cements increase with respect to the control cement. X-ray diffraction analysis, mechanical tests, and SEM investigations were carried out on the cements after different times of soaking in physiological solution. The rate of transformation of alpha-TCP into calcium deficient hydroxyapatite, as well as the microstructure of the cements, is not affected by the presence of Alendronate and Pamidronate. At variance, the bisphosphonates provoke a modest worsening of the mechanical properties. MG63 osteoblasts grown on the cements show a normal morphology and biological tests demonstrate very good rate of proliferation and viability in every experimental time. In particular, both Alendronate and Pamidronate promote osteoblast proliferation and differentiation, whereas they inhibit osteoclastogenesis and osteoclast function.

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+).

Porous composite scaffolds based on gelatin and partially hydrolyzed alpha-tricalcium phosphate.

Porous composite scaffolds of varying compositions were prepared by freeze-drying gelatin foams containing increasing amounts of alpha-tricalcium phosphate (alpha-TCP), up to about 40 wt.%. Due to the presence of gelatin, a partial hydrolysis of alpha-TCP into octacalcium phosphate (OCP) occurs during foaming. As a consequence, the scaffolds contain both alpha-TCP and OCP, in relative amounts of about 74% and 26%, respectively, independent of the initial composition. In physiological conditions the inorganic component of the scaffolds undergoes a further hydrolysis as shown by the finding that after immersion in phosphate-buffered saline at 37 degrees C for 1 week the scaffolds contain poorly crystalline hydroxyapatite together with OCP. The scaffolds display a porous interconnected microstructure. The mean dimensions of the pores decrease from about 350 to about 170 microm as the inorganic phase content increases. Simultaneously, the mean values of the compression strength and Young's modulus increase. Stabilization of the scaffolds was obtained by using a natural, non-toxic, crosslinking agent, genipin, which significantly improves their mechanical properties.

Setting properties and in vitro bioactivity of strontium-enriched gelatin-calcium phosphate bone cements.

Strontium is known to reduce bone resorption and stimulate bone formation. We have investigated the effect of strontium on the setting properties and in vitro bioactivity of a biomimetic gelatin-calcium phosphate bone cement. Gelatin-alpha-TCP powders, with a gelatin content of 15 wt %, were prepared by grinding and sieving the solid compounds obtained by casting gelatin aqueous solutions containing alpha-TCP. 5 wt % of CaHPO(4).2H(2)O were added to the cement powders before mixing with the liquid phase, with a L/P ratio of 0.3 mL/g. Strontium was added as SrCl(2).6H(2)O in different amounts up to 5 atom %. X-ray diffraction analysis, mechanical tests, and SEM investigations were carried out on the cements after different times of soaking in physiological solution. The presence of strontium affects both the initial and the final setting times of the cements, which increase with the ion content. The microstructural modifications observed in the SEM micrographs of the fractured surfaces are in agreement with the increase of the total porosity, and with the slight reduction of the compressive strength of the aged cements, on increasing strontium content. The rate of transformation of alpha-TCP into calcium deficient hydroxyapatite increases on increasing strontium content. SEM reveals that MG63 osteoblasts grown on the cements show a normal morphology and biological tests demonstrate very good rate of proliferation and viability in every experimental time. In particular, strontium stimulates Alkaline Phosphatase activity, Collagen type I, osteocalcin, and osteoprotegerin expression.

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 mineralization of gelatin-polyacrylic acid complex matrices.

Gelatin-polyacrylic acid (gel-PAA) matrices were obtained by slow diffusion of polyacrylic acid into gelatin gels. The matrices were submitted to uniaxial stretching, which induces a preferential orientation of the collagen molecules, and used as biomimetic substrates for the nucleation of hydroxyapatite from simulated body fluid (SBF). The relative amount of hydroxyapatite deposited from 1.5SBF increases as a function of polyelectrolyte content in the matrices, up to about 30 wt%. In the absence of PAA, the inorganic phase is laid down on the surface of the gelatin matrices as hemispherical aggregates. At variance, hydroxyapatite deposition in the gel-PAA composite matrices at relatively low PAA content occurs preferentially in the spaces between the layers on the surface of the matrices and displays a tablet-like morphology. At high polyelectrolyte concentration, an almost uniform layer of hydroxyapatite covers the whole surface of the matrices. The preferential orientation of the (002) hydroxyapatite reflection indicates a close relationship between the inorganic crystals and the collagen molecules.