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.

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.

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.

Size-segregated aerosol in a hot-spot pollution urban area: Chemical composition and three-way source apportionment.

In this work, a comprehensive characterisation and source apportionment of size-segregated aerosol collected using a multistage cascade impactor was performed. The samples were collected during wintertime in Milan (Italy), which is located in the Po Valley, one of the main pollution hot-spot areas in Europe. For every sampling, size-segregated mass concentration, elemental and ionic composition, and levoglucosan concentration were determined. Size-segregated data were inverted using the program MICRON to identify and quantify modal contributions of all the measured components. The detailed chemical characterisation allowed the application of a three-way (3-D) receptor model (implemented using Multilinear Engine) for size-segregated source apportionment and chemical profiles identification. It is noteworthy that - as far as we know - this is the first time that three-way source apportionment is attempted using data of aerosol collected by traditional cascade impactors. Seven factors were identified: wood burning, industry, resuspended dust, regional aerosol, construction works, traffic 1, and traffic 2. Further insights into size-segregated factor profiles suggested that the traffic 1 factor can be associated to diesel vehicles and traffic 2 to gasoline vehicles. The regional aerosol factor resulted to be the main contributor (nearly 50%) to the droplet mode (accumulation sub-mode with modal diameter in the range 0.5-1 µm), whereas the overall contribution from the two factors related to traffic was the most important one in the other size modes (34-41%). The results showed that applying a 3-D receptor model to size-segregated samples allows identifying factors of local and regional origin while receptor modelling on integrated PM fractions usually singles out factors characterised by primary (e.g. industry, traffic, soil dust) and secondary (e.g. ammonium sulphate and nitrate) origin. Furthermore, the results suggested that the information on size-segregated chemical composition in different size classes was exploited by the model to relate primary emissions to rapidly-formed secondary compounds.

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.

Tracheal rhinoscleroma.

INTRODUCTION: Tracheal rhinoscleroma is an infectious granulomatosis of the tracheobronchial tract caused by a Gram-negative bacillus. Exclusively tracheal involvement has been rarely reported in the literature. The purpose of this study was to report a case of subglottic stenosis secondary to rhinoscleroma. SUMMARY: A 46-year-old North African woman with no medical or surgical history presented with inspiratory dyspnoea that had been present for several years. Endoscopic examination under general anaesthesia revealed tracheal stenosis. Histological examination of mucosal biopsies demonstrated Mikulicz cells and culture of bacteriological samples taken during a second biopsy confirmed the diagnosis of rhinoscleroma. CO2 laser subglottic obstruction relief was performed and treatment with ofloxacin was initiated. No recurrence of tracheal stenosis was observed with a follow-up of 6 months. DISCUSSION: The diagnosis of rhinoscleroma is based on histological and bacteriological examination. Cultures are positive in 60% of cases, but negative cultures do not exclude the diagnosis of rhinoscleroma. Specific treatment consists of long-term antibiotic therapy, while surgery may be indicated for symptomatic treatment.

Cyclophilin D counteracts P53-mediated growth arrest and promotes Ras tumorigenesis.

Mitochondrial alterations induced by oncogenes are known to be crucial for tumorigenesis. Ras oncogene leads to proliferative signals through a Raf-1/MEK/ERK kinase cascade, whose components have been found to be also associated with mitochondria. The mitochondrial pepdidyl-prolyl isomerase cyclophilin D (CypD) is an important regulator of the mitochondrial permeability transition and a key player in mitochondria physiology; however, its role in cancer is still unclear. Using cellular and in vivo mouse models, we demonstrated that CypD protein upregulation induced by oncogenic Ras through the Raf-1/MEK/ERK pathway has a deterministic role in tumor progression. In fact, targeting CypD gene expression clearly affected RasV12-induced transformation, as showed by in vitro data on murine NIH3T3 and human MCF10A mammary epithelial cells. In addition, studies in xenograft and K-Ras lung cancer mouse models demonstrated that genetic deletion or pharmacological suppression of CypD efficiently prevented Ras-dependent tumor formation. Furthermore, Erbb2-mediated breast tumorigenesis was similarly prevented by targeting CypD. From a mechanistic point of view, CypD expression was associated with a reduced induction of p21(WAF1/CIP1) and p53 functions, unraveling an antagonistic function of CypD on p21-p53-mediated growth suppression. CypD activity is p53 dependent. Interestingly, a physical association between p53 and CypD was detected in mitochondria of MCF10A cells; furthermore, both in vitro and in vivo studies proved that CypD inhibitor-based treatment was able to efficiently impair this interaction, leading to a tumor formation reduction. All together, these findings indicate that the countering effect of CypD on the p53-p21 pathway participates in oncogene-dependent transformation.

X-ray diffraction study of bovine lens capsule collagen.

The wide angle X-ray diffraction pattern of air-dried lens capsule collagen under tension is the same as the tendon collagen diffraction pattern with regard to the main reflections, and indicates that lens capsule collagen has the characteristic three-stranded helical structure with an axial repeat of 0.29 nm as tendon collagen. The low angle X-ray diffraction pattern shows several weak diffraction maxima corresponding to the meridional reflections of capsule collagen which show orders of 63.0 nm periodicity. This is an evidence of quarter staggered molecular assembly typical of tendon collagen even if less ordered. The results are consistent with the existence in lens capsule collagen of clearly defined molecular units, which can be oriented by stress and are packed in a poor-ordered fibrillar assembly.

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.

Surgical treatment of a case of adult epiglottic laryngomalacia.

INTRODUCTION: Adult laryngomalacia is rare. It may be idiopathic or secondary to trauma or degenerative disease. CASE REPORT: A 25-year-old man presented with inspiratory dyspnea on effort of several months' evolution. Flexible endoscopy found epiglottic laryngomalacia, managed by CO2 laser V-shaped partial epiglottectomy. DISCUSSION: Excessive resection of the epiglottis may lead to false passage; insufficient resection risks being ineffective. V-shaped partial epiglottectomy minimizes risk of false passage while ensuring permanent respiratory airflow via the epiglottic V during epiglottic movement.

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.

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.

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.

Hydroxyapatite-gelatin films: a structural and mechanical characterization.

Composite films of gelatin and hydroxyapatite were prepared and characterized by mechanical tests, scanning electron microscopy and X-ray diffraction investigation. The mechanical properties of the films are greatly affected by the presence of hydroxyapatite and change as a function of inorganic phase content. On stretching, the long axis of the collagen molecular portions align parallel to the direction of deformation and the gelatin coarse layered structure becomes more evident and ordered. Furthermore, under deformation the inorganic crystals, which are embedded in the gelatin layers, seem to squeeze out in the interlayer spaces and assume a preferential orientation parallel to the force trajectories. Thus, as the inorganic phase stiffens the gelatin films, the macromolecular matrix distributes the stress promoting the preferential orientation of the apatitic crystals. The results indicate that this experimental approach can be used to prepare composites with anisotropic properties, which can be modulated through variation in composition and mechanical deformation in order to get biomaterials suitable to fulfill specific mechanical functions.

In vitro calcified tendon collagen: an atomic force and scanning electron microscopy investigation.

Atomic force microscopy (AFM), scanning electron microscopy and X-ray energy dispersive spectroscopy have been performed on decalcified turkey tendons submitted to in vitro calcification in order to investigate the morphology and the surface relationships between the inorganic phase and the collagen fibres during deposition and compare with those found for physiologically calcified samples. 'Tapping mode' AFM was used to reduce the vertical force applied to the samples, which were examined without any preparation. A further characterization has been carried out by means of X-ray diffraction, infrared absorption and chemical analyses. The observations indicate that the inorganic phase deposited on collagen fibres during in vitro calcification is poorly crystalline B carbonated apatite. The composition, structure and dimensions of apatitic crystallites, as well as their orientation with respect to collagen fibrils, are very similar to those characteristic of physiologically calcified tissues. However, the crystallites seem to be nucleated on the fibril surface, without appreciably affecting the molecular packing of collagen.

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.

X-ray diffraction study of in vitro calcification of tendon collagen.

Decalcified samples of turkey leg tendon were submitted to in vitro calcification in the presence of metastable solutions of calcium phosphate at different concentrations. The structural relationship between apatitic deposits and collagen fibrils was examined by high- and small-angle X-ray diffraction using conventional and synchrotron radiation sources. At high supersaturation the apatitic crystallites were deposited on the collagen fibrils with their crystallographic c-axis preferentially oriented parallel to the fibril axis. At lower supersaturation, a fraction of the apatitic crystallites also grew with the c-axis preferentially oriented parallel to the collagen fibril axis, whereas other exhibited a preferential orientation perpendicular to the fibril axis. The analysis of the small-angle X-ray diffraction data indicates that the deposition of the apatitic phase in the sample stored in solution at lower supersaturation induced modifications of the collagen electron density distribution in the axial direction, which can be attributed to the deposition of the inorganic crystallites inside the gap region of the collagen structure.

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.

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.

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.