Adriana Bigi

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.

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.

Electrospun gelatin nanofibers: Optimization of genipin cross-linking to preserve fiber morphology after exposure to water

The development of suitable biomimetic three-dimensional scaffolds is a fundamental requirement of tissue engineering. This paper presents the first successful attempt to obtain electrospun gelatin nanofibers cross-linked with a low toxicity agent, genipin, and able to retain the original nanofiber morphology after water exposure. The optimized procedure involves an electrospinning solution containing 30 wt.% gelatin in 60/40 acetic acid/water (v/v) and a small amount of genipin, followed by further cross-linking of the as-electrospun mats in 5% genipin solution for 7 days, rinsing in phosphate-buffered saline and then air drying at 37°C. The results of scanning electron microscopy investigations indicated that the cross-linked nanofibers were defect free and very regular and they also maintained the original morphology after exposure to water. Genipin addition to the electrospinning solution dramatically reduced the extensibility of the as-electrospun mats, which displayed further remarkable improvements in elastic modulus and stress at break after successive cross-linking up to values of about 990 and 21 MPa, respectively. The results of the preliminary in vitro tests carried out using vascular wall mesenchymal stem cells indicated good cell viability and adhesion to the gelatin scaffolds.

Effect of Mg2+, Sr2+, and Mn2+ 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+).

Isomorphous substitutions in β-tricalcium phosphate: The different effects of zinc and strontium

Abstract X-ray diffraction and infrared absorption analyses have been carried out on zinc-substituted and strontium-substituted β-tricalcium phosphate prepared by solid-state reaction. Zinc can substitute calcium up to 20 atom %, inducing a nonlinear variation of the lattice constants and an increase in degeneracy of the PO 4 3− infrared absorption bands. On the other hand, up to 80 atom % of strontium can enter into the crystal structure of β-tricalcium phosphate, causing a linear enlargement of the unit cell, in agreement with its greater ionic radius compared to that of calcium. Furthermore, strontium incorporation provokes the shift of the PO 4 3− absorption bands toward lower frequencies. On the basis of the data previously obtained on magnesium-substituted β-tricalcium phosphate, the different behaviours exhibited by zinc and strontium could be attributed to a different distribution into the cationic sites of the β-tricalcium phosphate structure. The results allow us to relate the effect of bivalent ions on the structure and relative stability of calcium phosphates with their ionic radius, and can be utilized to interpret the role of ionic composition on the properties of biological phosphates.

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.

Effects of freezing on the biomechanical and structural properties of human posterior tibial tendons

This work analyzes the effects of storage by fresh-freezing at −80°C on the histological, structural and biomechanical properties of the human posterior tibial tendon (PTT), used for ACL reconstruction. Twenty-two PTTs were harvested from eleven donors. For each donor one tendon was frozen at −80°C and thawed in physiological solution at 37°C, and the other was tested without freezing (control). Transmission electron microscopy (TEM), differential scanning calorimetry (DSC) and biomechanical analysis were performed. We found the following mean changes in frozen-thawed tendons compared to controls: TEM showed an increase in the mean diameter of collagen fibrils and in fibril non-occupation mean ratio, while the mean number of fibrils decreased; DSC showed a decrease in mean denaturation temperature and denaturation enthalpy. Biomechanical analysis showed a decrease in ultimate load and ultimate stress, an increase in stiffness and a decrease in ultimate strain of tendons. In conclusion fresh-freezing brings about significant changes in the biomechanical and structural properties of the human PTT. A high variability exists in the biophysical properties of tendons among individuals and in the effects of storage on tendons. Therefore, when choosing an allograft tendon, particular care is needed to choose a biomechanically suitable graft.RésuméCe travail a pour but d’analyser les effets du stockage à −80°C sur le plan histologique, structurel et biomécanique d’un tendon le tibial ou jambier postérieur (PTT), utilisé pour la reconstruction des ligaments croisés antérieurs. 22 PTT ont été conservées provenant de 11 donneurs. Pour chaque donneur un tendon a été congelé à −80°C et l’autre, conservé dans une solution physiologique à 37°C. Ces tendons ont été testés. L’examen par microscope électronique (TEM), le scanner calorimétrique (DSC) et une analyse biomécanique ont été réalisés. Nous avons trouvé des changements dans les tendons conservés au froid en comparaison du groupe contrôle. Le TEM, examen au microscope électronique a montré une diminution du diamètre des fibres collagènes. L’analyse biomécanique a montré également une diminution de la résistance à la charge et au stress ainsi qu’une augmentation de la rigidité et une diminution des contraintes terminales au niveau du tendon. En conclusion: la congélation des tendons frais amène des modifications significatives des caractéristiques biomécaniques et structurelles du tendon PTT humain. Il existe une variation importante des propriétés biophysiques des tendons parmi les individus et du fait de leurs conservations. Pour cela, il est nécessaire lorsque l’on choisit un tendon et une allogreffe du tendon d’apporter dans le choix sur le plan biomécanique un soin particulier.

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 Youngs 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.

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.

Biofunctional alendronate-Hydroxyapatite thin films deposited by Matrix Assisted Pulsed Laser Evaporation

We applied Matrix Assisted Pulsed Laser Evaporation (MAPLE) in order to synthesize alendronate-hydroxyapatite thin films on titanium substrates. Alendronate-hydroxyapatite composite nanocrystals with increasing bisphosphonate content (0, 3.9, 7.1%wt) were synthesized in aqueous medium. Then, they were suspended in deionised water, frozen at liquid nitrogen temperature and used as targets for MAPLE experiments. The depositions were conducted with a KrF* excimer laser source (l=248nm, t(FWHM)=25ns) in mild conditions of temperature and pressure. The obtained thin films had a good crystallinity, which slightly decreases with the increase of alendronate content, and exhibited a porous-like structure. Osteoblast-like MG63 cells and human osteoclasts were cultured on the thin films up to 14 days. In the presence of alendronate, MG63 cells displayed a normal morphology, increased proliferation and higher values of differentiation parameters, namely type I collagen, osteocalcin, and osteoprotegerin/TNF-related activation-induced cytokine receptor ratio. In contrast, osteoclasts showed significantly reduced proliferation, and increased level of Caspase 3. Moreover, the coatings synthesized from hydroxyapatite at relatively high bisphosphonate content (7.1% wt) displayed a reduced production of Tumour Necrosis Factor alpha (TNF-alpha) and Interleukin 6 (IL-6), suggesting a down-regulatory role of alendronate on the inflammatory reaction. The successful deposition of alendronate modified hydroxyapatite thin films yields coatings with enhanced bioactivity, able to promote osteoblast differentiation and to inhibit osteoclast proliferation.