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.

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.

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+).

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.

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.

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.

Normal and osteopenic bone-derived osteoblast response to a biomimetic gelatin-calcium phosphate bone cement.

We have recently developed a new calcium phosphate bone cement enriched with gelatin (GEL-CP), which exhibits improved mechanical properties with respect to the control cement (C-CP). In a previous study, we demonstrated the good response of osteoblast-like cells to the new biomimetic bone cement. Herein, we extend the investigation to primary culture of osteoblasts derived from healthy and pathological bones. Osteoblasts derived from normal (N-OB) and osteopenic (O-OB) sheep bones were cultured on samples of GEL-CP, and their behavior was compared with that of cells cultured on C-CP as control. Cell morphology, proliferation, and differentiation were evaluated at 3 and 7 days. SEM analysis revealed that both N-OB and O-OB showed a normal morphology when cultured on GEL-CP. Biological tests demonstrated that the gelatin-enriched cement improves osteoblasts' activity and differentiation of O-OB cultures, with respect to the control samples. The data indicate that the new composite cement positively stimulates alkaline phosphatase activity, collagen type I, and osteocalcin production, not only in N-OB, but also in O-OB culture. The improvement due to the presence of gelatin suggests that the biomimetic composite material could be successfully applied as bone substitute also in the presence of osteopenic bone.

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.

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.

Relationship between triple-helix content and mechanical properties of gelatin films.

This paper reports a study on the influence of the renaturation level of gelatin on the mechanical and swelling properties of gelatin films. Films at different renaturation level were obtained from gelatin samples with different Bloom index. It was verified that the triple-helix content, calculated from the values of the enthalpy of denaturation associated to the endothermal transition at about 41 degrees C of gelatin, increases with the Bloom index. The d.s.c. data are further supported by the results of the X-ray diffraction investigation carried out on the same samples. The increase of triple-helix content provokes a significant reduction in the degree of swelling, and a remarkable improvement of the mechanical properties of the films. The elastic Young's modulus, E, increases linearly with the renaturation level, from 3.6 to 12.0 MPa. Crosslinking with GTA 1% remarkably reduces the degree of swelling of all the samples, and induces a further increase of the Young's modulus, which reaches values up to 27 MPa.

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.

Alpha-Tricalcium phosphate-gelatin composite cements.

This study investigates gelatin influence on calcium phosphate cement (CPC) setting properties. Cements of different compositions were prepared using |Alpha -tricalcium phosphate (|Alpha -TCP) enriched with a small amount of dicalcium phosphate dihydrate (DCPD), and different gelatin amounts up to 20 wt%. The cements, prepared with a liquid/powder ratio of 0.3 ml/g, were soaked in simulated body fluid (SBF) for different times inverted exclamation markU21 days. The setting reaction of the control cement prepared without gelatin occurred in 7 days, whereas the transformation of the cements at high gelatin content into apatite was almost complete in 2-day aging in SBF. Gelatin presence reduced the total porosity of the cements, and significantly modified their microstructure. The fractured surface of the aged control cement was covered with entangled plate-like apatite crystals, whereas the gelatin cements displayed more compact surfaces, most likely due to the inhibiting effect of gelatin on apatite crystal growth. The microstructural modifications were in agreement with the mprovement of the mechanical properties in the aged cements, the compressive strength of which increased linearly as a function of gelatin content, from 2.0 inverted exclamation markA 0.8 to 14 inverted exclamation markA 1 MPa.

Porous phosphate-gelatine composite as bone graft with drug delivery function.

The design and synthesis of porous phosphate-gelatine composite implant which mimicks the structure of natural bone and has drug delivery function is proposed. Gelatine reproducing the proteinaceous part of bone was cross-linked in order to modulate its solubility in the physiologic fluids. The kinetic of gelatine release from ceramic matrix was also evaluated as model of the release of any therapeutic compound which can be loaded into gelatine.

Stabilization of gelatin films by crosslinking with genipin.

The possibility to stabilize gelatin films by crosslinking with genipin was investigated through a mechanical, chemical and thermal characterization of samples treated with genipin solutions at different concentrations. The extent of crosslinking, evaluated as difference between the number of free epsilon -amino groups before and after crosslinking, increases as a function of genipin concentration up to about 85%. Simultaneously, the deformability of the films decreases whereas the Young's modulus E, increases. Furthermore, crosslinking provokes a significant reduction of the swelling in physiological solution, and enhances the thermal stability of the samples, as indicated by the results of the d.s.c. investigation. The data obtained from the films treated with genipin at concentrations higher than 0.67% are quite similar, and indicative of a good stabilizing effect of genipin. In spite of the small gelatin release (2%) observed after 1 month of storage in buffer solution, the mechanical, thermal and swelling properties of the films are very close to those previously obtained for glutaraldehyde crosslinked gelatin, and suggest that genipin, which is by far less cytotoxic, can be considered a valid alternative for crosslinking gelatin biomaterials.

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.

Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking.

The mechanical, thermal, swelling and release properties of glutaraldehyde (GTA) crosslinked gelatin films have been investigated in order to verify the influence of GTA concentration on the stability of the films. Air-dried films were submitted to treatment with GTA solutions at concentrations ranging from 0.05 to 2.5 wt%. At the smallest GTA concentration, the crosslinking degree, determined by trinitrobenzensulfonic acid assay, amounts to about 60% and increases up to values near 100%, obtained with GTA concentrations > or = 1 wt%. Simultaneously, the deformability of the films decreases, whereas the stress at break, sigmab, and the Young's modulus, E, increase. A crosslinking degree of about 85%, obtained using 0.25% GTA, is enough to prevent gelatin release in buffer solution and to provoke a significant reduction of the swelling in physiological solution. Furthermore, crosslinking greatly affects the thermal stability of the samples, as indicated by the results of differential scanning calorimetry (d.s.c.) investigation carried out on wet and air-dried films. The data suggest that the use of GTA at low concentration, which is desiderable to prevent toxicity, allows to modulate the physico-chemical properties of gelatin films, in order to obtain stable materials with a wide range of possible biomedical applications.