Solvent and melting induced microspheres sintering techniques: a comparative study of morphology and mechanical properties.

In this work we propose a bottom up approach founded on the assembly of building blocks by solvent induced microparticle sintering to realize multifunctional polymer scaffolds with predefined pore dimension and fully percolative pathway, able to include interspersing microdepot for the release of bioactive molecules. The aim of this study was to develop a versatile method of microspheres sintering based on the partial dissolution of the surface of adjacent microparticles and to compare it with melting induced microspheres sintering, just developed in a previous work. The two techniques were compared in terms of morphology, porosity and mechanical properties. The high potential of customizing the sintering process by the proper selection of the sintering techniques as well as microparticles with different features (e.g., material, size, shape, inner porosity) allows obtaining a wide pattern of micro/nanostructures with bio-inspired mechanical response so satisfying all basic requirements of a "smart" scaffold for bone tissue engineering.

Optimizing PANi doped electroactive substrates as patches for the regeneration of cardiac muscle.

In scaffold aided regeneration of muscular tissue, composite materials are currently utilized as a temporary substrate to stimulate tissue formation by controlled electrochemical signals as well as continuous mechanical stimulation until the regeneration processes are completed. Among them, composites from the blending of conductive (CPs) and biocompatible polymers are powerfully emerging as a successful strategy for the regeneration of myocardium due to their unique conductive and biological recognition properties able to assure a more efficient electroactive stimulation of cells. Here, different composite substrates made of synthesized polyaniline (sPANi) and polycaprolactone (PCL) were investigated as platforms for cardiac tissue regeneration. Preliminary, a comparative analysis of substrates conductivity performed on casted films endowed with synthesized polyaniline (sPANi) short fibres or blended with emeraldine base polyaniline (EBPANi) allows to study the attitude of charge transport, depending on the conducting filler amount, shape and spatial distribution. In particular, conducibility tests indicated that sPANi short fibres provide a more efficient transfer of electric signal due to the spatial organization of electroactive needle-like phases up to form a percolative network. On the basis of this characterization, sPANi/PCL electrospun membranes have been also optimized to mimic either the morphological and functional features of the cardiac muscle ECM. The presence of sPANi does not relevantly affect the fibre architecture as confirmed by SEM/image analysis investigation which shows a broader distribution of fibres with only a slight reduction of the average fibre diameter from 7.1 to 6.4 µm. Meanwhile, biological assays--evaluation of cell survival rate by MTT assay and immunostaining of sarcomeric α-actinin of cardiomyocites-like cells--clearly indicate that conductive signals offered by PANi needles, promote the cardiogenic differentiation of hMSC into cardiomyocite-like cells. These preliminary results concur to promise the development of electroactive biodegradable substrates able to efficiently stimulate the basic cell mechanisms, paving the way towards a new generation of synthetic patches for the support of the regeneration of damaged myocardium.

Synergistic effect of sericin and keratin in gelatin based nanofibers for in vitro applications.

Protein-based nanomaterials are gaining growing interest in biomedical field. The present paper evaluates the physico-chemical properties of electrospun nanofibers resulting from the combination of gelatin with keratin (from wool) and sericin (from silk) to validate their use for in vitro interaction studies. We demonstrated that that presence of sericin influences the fiber morphology at macroscopic level - i.e., wide diameter distributions by SEM and image analysis - with effects on chemical - i.e., a decrease of hydrogen bonds of NH groups verified by infrared spectroscopy - and thermal behavior of electrospun nanofibers, in comparison with gelatin-based ones. Moreover, we verified that sericin, in combination with keratin macromolecules, can amplify the biochemical signal of gelatin, improving the in-vitro stability of gelatin-based nanofibers. In vitro results confirm a synergistic effect of sericin and keratin on human Mesenchymal Stem Cells (hMSC) proliferation - increase over 50% respect to other types - associated to the enhancement of in vitro stability directly ascribable to the peculiar physical interaction among the proteins. These findings suggest the use of sericin/keratin/gelatin enriched electrospun fibers as nanostructured platforms for interface tissue engineering.

Temperature-driven processing techniques for manufacturing fully interconnected porous scaffolds in bone tissue engineering.

The development of structures with a predefined multiscale pore network is a major challenge in designing tissue engineering (TE) scaffolds. To address this, several strategies have been investigated to provide biocompatible, biodegradable porous materials that would be suitable for use as scaffolds, and able to guide and facilitate the cell activity involved in the generation of new tissue regeneration. This study seeks to provide an overview of different temperature-driven process technologies for developing scaffolds with tailored porosity, in which pore size distribution is strictly defined and pores are fully interconnected. Here, three-dimensional (3D) porous composite scaffolds based on poly(epsilon-caprolactone) (PCL) were fabricated by thermally induced phase separation (TIPS) and by melt co-continuous polymer blending (MCPB). The combination of these processes with a salt leaching technique enables the establishment of bimodal porosity within the polymer network. This feature may be exploited in the development of substrates with fully interconnected pores, which can be used effectively for tissue regeneration. Various combinations of the proposed techniques provide a range of procedures for the preparation of porous scaffolds with an appropriate combination of morphological and mechanical properties to reproduce the requisite features of the extracellular matrix (ECM) of hard tissues such as bone.

The performance of poly-epsilon-caprolactone scaffolds in a rabbit femur model with and without autologous stromal cells and BMP4.

The ability of a cellular construct to guide and promote tissue repair strongly relies on three components, namely, cell, scaffold and growth factors. We aimed to investigate the osteopromotive properties of cellular constructs composed of poly-epsilon-caprolactone (PCL) and rabbit bone marrow stromal cells (BMSCs), or BMSCs engineered to express bone morphogenetic protein 4 (BMP4). Highly porous biodegradable PCL scaffolds were obtained via phase inversion/salt leaching technique. BMSCs and transfected BMSCs were seeded within the scaffolds by using an alternate flow perfusion system and implanted into non-critical size defects in New Zealand rabbit femurs. In vivo biocompatibility, osteogenic and angiogenic effects induced by the presence of scaffolds were assessed by histology and histomorphometry of the femurs, retrieved 4 and 8 weeks after surgery. PCL without cells showed scarce bone formation at the scaffold-bone interface (29% bone/implant contact and 62% fibrous tissue/implant contact) and scarce PCL resorption (16%). Conversely, PCL seeded with autologous BMSCs stimulated new tissue formation into the macropores of the implant (20%) and neo-tissue vascularization. Finally, the BMP4-expressing BMSCs strongly favoured osteoinductivity of cellular constructs, as demonstrated by a more extensive bone/scaffold contact.

Porosity and mechanical properties relationship in PCL porous scaffolds.

Scaffold design plays a pivotal role in tissue engineering and regenerative medicine approaches for creating biological alternatives for implants. The crucial aspect in scaffold design consists of the development of highly porous scaffolds, with strict control of porosity features (porosity degree and pore sizes), continuing to provide an adequate mechanical response, mainly in compressive loading, both in vitro and in vivo conditions. A study was undertaken of three-dimensional (3D) porous scaffolds obtained from poly epsilon-caprolactone solution through the phase inversion/salt leaching technique. In particular, the influence of structural porosity features on mechanical response was investigated to establish the correlation between structural parameters and compressive response. Scaffold porosity features can be controlled by changing the amount and size of the porogen agent used. Mechanical response in compression is consistent with porosity features: elastic modulus calculated in the toe region range (0-0.1 of total strain) shows an increase from 0.24-1.85 MPa coherently, with a reduction in pore volume fraction from 84.9 to 45.7%. Such behavior can be predicted by using analytical models for the determination of the elastic modulus of cellular solids based on the morphological assumption of cubic cell structure (cubic open cell (COC) and cubic closed cells (CCC)). Compressive behavior prediction offered by the proposed models is in agreement with the experimental results in the case of higher pore volume fractions according to the theoretical results of other investigators.

HepG2 and human healthy hepatocyte in vitro culture and co-culture in PCL electrospun platforms.

The discovery of new drugs to treat pathological cells in the case of aggressive liver primary cancer is imposing the identification of high-throughput screening systems to predict the in vivo response of new therapeutic molecules, in order to reduce current use of animals and drug testing costs. Recently, micro/nanostructured scaffolds have been adopted to reproduce the hepatic microenvironment due to their higher similarity to the biological niche with respect to the traditional two-dimensional culture plate, so providing novel in vitro models for reliably understanding molecular mechanisms related to cancer cells activity. Herein, we propose the study of electrospun scaffolds made of polycaprolactone as in vitro model that can mimic the morphological organization of native extracellular matrix and the co-culture of hepatic cell lines-i.e., HepG2, human healthy hepatocytes (HHH). The micro- and nano-scale morphological features of fibers with diameter equal to (3.22 ± 0.42) µm and surface roughness of (17.84 ± 4.43) nm-allow the reproduction of the in vivo scenario influencing the adhesion and proliferation rate of the cultured cells. A much lower proliferation rate is observed for the HepG2 cells compared to the HHH cells, when cultured on the fibrous scaffolds over a time course of 4 weeks. Moreover, results on oxidative stress mechanisms indicate an antioxidant effect of fibers mainly in the case of co-colture, thus suggesting a promising use as new in vitro models to explore alternative therapeutic strategies in hepatocarcinoma treatment.

Needle-like ion-doped hydroxyapatite crystals influence osteogenic properties of PCL composite scaffolds.

Surface topography and chemistry both play a crucial role on influencing cell response in 3D porous scaffolds in terms of osteogenesis. Inorganic materials with peculiar morphology and chemical functionalities may be proficiently used to improve scaffold properties-in the bulk and along pore surface-promoting in vitro and in vivo osseous tissue in-growth. The present study is aimed at investigating how bone regenerative properties of composite scaffolds made of poly(Ɛ-caprolactone) (PCL) can be augmented by the peculiar properties of Mg(2+) ion doped hydroxyapatite (dHA) crystals, mainly emphasizing the role of crystal shape on cell activities mediated by microstructural properties. At the first stage, the study of mechanical response by crossing experimental compression tests and theoretical simulation via empirical models, allow recognizing a significant contribution of dHA shape factor on scaffold elastic moduli variation as a function of the relative volume fraction. Secondly, the peculiar needle-like shape of dHA crystals also influences microscopic (i.e. crystallinity, adhesion forces) and macroscopic (i.e. roughness) properties with relevant effects on biological response of the composite scaffold: differential scanning calorimetry (DSC) analyses clearly indicate a reduction of crystallization heat-from 66.75 to 43.05 J g(-1)-while atomic force microscopy (AFM) ones show a significant increase of roughness-from (78.15 ± 32.71) to (136.13 ± 63.21) nm-and of pull-off forces-from 33.7% to 48.7%. Accordingly, experimental studies with MG-63 osteoblast-like cells show a more efficient in vitro secretion of alkaline phosphatase (ALP) and collagen I and a more copious in vivo formation of new bone trabeculae, thus suggesting a relevant role of dHA to support the main mechanisms involved in bone regeneration.

The automated multiparameter analyzer for cells (AMAC) IIA, a true bridge circuit Coulter-type electronic cell volume transducer.

A true bridge Coulter effect (electronic cell volume) transducer has been developed. All resistances of this bridge are now the result of current flow through saline channels. Contamination by electrode products including gas bubbles has been completely eliminated since both power electrodes are now remote from the flow chamber. Since the orifice is in series with an approximately 10 K ohm resistance generated by a gel-filled capillary and a displacement rheostat, it floats electrically, at virtual ground. The other side of the bridge consists of a fluid side-wire. Removing the power electrode from the orifice outlet makes possible downward flow and the use of a single outer sheath, and eliminates noise generated by gas bubbles which could possibly be trapped. It should now be possible to combine this design with that of the AMAC III square orifice, to produce an electro-optical sorter where all parameters are measured simultaneously. This true bridge circuit possesses the further advantage that noise due both to the power supply and to overvoltage at the power electrodes is common-mode rejected, and any drift due to changes in electrode polarization is eliminated. Preliminary experiments confirm results with the AMAC II that hemoglobinopathies can be recognized by the increased coefficient of variation (CV) of the erythrocyte spectra.

Organ tolerance of non-steroidal anti-inflammatory drugs: effect on liver cells.

Experimental studies in mice and in rats showed the good tolerance of a new non-steroidal anti-inflammatory drug, flunoxaprofen, by normal and CCl4 damaged liver. The activity of an enzyme-inducing drug, such as phenobarbital, showed a greater increase after pretreatment with indomethacin than with flunoxaprofen or benoxaprofen. High doses of benoxaprofen and indomethacin significantly decreased bromosulphonphthalein excretion in rats with normal or CCl4 damaged liver; the effect was not observed with flunoxaprofen administered at the same dose as benoxaprofen. Moreover, benoxaprofen and indomethacin but not flunoxaprofen induced a significant increase of some serum liver enzymes in CCl4 poisoned rats.

Centrifugal cytology of nipple aspirate cells.

Two improvements in the methodology for obtaining and preparing nipple aspirates from nonlactating women are reported. The first is the development and use of a new breast pump with a controllable vacuum and cups of various sizes. The second is the use of centrifugal cytology to prepare the dispersions. Twenty-one of 24 breasts of patients in the age range 30 to 49 years produced cellular dispersions which contained foam cells; of them, 13 contained ductal cells. A comparison of glutaraldehyde and ethanol fixation indicated that the cells appeared substantially the same.

[The portal venous bed]. / A proposito del letto venoso portale.

The AA. give description of the anatomical bases of the venous system of compensation in the syndrome of portal hypertension giving iconographic pictures. They describe the major characteristic features of the portal vein circulation with particular attention to the main variations, their forms, site and particularly with reference to the surgical operations they necessitate.

Evaluation of DOWNSTAGING as leading concept in sphincter-saving surgery for rectal cancer after preoperative radio-chemotherapy (Preop RCT).

AIM: To evaluate the downstaging of rectal cancer after preop R +/- CT. METHODS: 392 patients (pts) with rectal cancer were observed. Only 172 pts (58%) with II and III stage cancer of middle and lower third were examined. Enrol-led pts were 168: 52 of them received preop R +/- CT (32 RT, 20 R + CT). Preop R +/- CT group included 14 middle third cancers (73%), 38 lower third (17%). In this group, tumor stage was as follows: 44 T3 stage tumors (86.4%), 8 (15.4%) T4. Mean age of this group was 57 years (range 42-67). Patients received 45 Gy for 5 weeks in 25 fractions and continuous administration of 5-FU (300-500 mg/m2/die). Surgery was performed 6 weeks +/- 7 days after the therapy. RESULTS: Downstaging, at least of 1 T-stage level, was detected in 45 patients (86%) (8 middle third; 32 lower third), in 5 (9.6%) (4 middle third, 1 lower third), tumor decreased to pT0N0, while in 7 (13.5%) (2 middle third, 5 lower third), there was no response. An Anterior Resection (AR) was performed in 40 patients (77%) [4 Downstaged to pT0N0 middle third cancers; 36 downstaged but with residual disease (8 middle third, 28 lower third)]; APR was performed in 12 (23%) (7 No responders patients, 1 Downstaged to pT0N0 lower third cancer, 4 downstaged but with residual disease of lower third). CONCLUSIONS: Preop R +/- CT is effective in obtaining a significative downstaging to allow sphincter saving surgery, without compromising oncological results.

Injectable calcium-phosphate-based composites for skeletal bone treatments.

Alpha-tricalcium-phosphate-based bone cements hydrolyze and set, producing calcium-deficient hydroxyapatite. They can result in an effective solution for bone defect reconstruction due to their biocompatibility, bioactivity and adaptation to shape and bone defect sizes, together with an excellent contact between bone and graft. Moreover, the integration of hydrogel phase based on poly(vinyl alcohol) (PVA) to H-cem-composed of α-tricalcium phosphate (98% wt) and hydroxyapatite (2% wt)-allows improving the mechanical and biological properties of the cement. The aim of this work was to evaluate the influence of the PVA on relevant properties for the final use of the injectable bone substitute, such as setting, hardening, injectability and in vivo behaviour. It was shown that by using PVA it is possible to modulate the setting and hardening properties: large increase in injectability time (1 h) in relation with the plain cement (few minutes) was achieved. Moreover, in vivo tests confirmed the ability of the composite to enhance bone healing in trabecular tissue. Histological results from critical size defects produced in rabbit distal femoral condyles showed after 12 weeks implantation a greater deposition of new tissue on bone-composite interfaces in comparison to bone-cement interfaces. The quality of bone growth was confirmed through histomorphometric and microhardness analysis. Bone formation in the composite implantation sites was significantly higher than in H-cem implants at both times of evaluation.

Biomineralized porous composite scaffolds prepared by chemical synthesis for bone tissue regeneration.

Scaffold design is a key factor in the clinical success of bone tissue engineering grafts. To date, no existing single biomaterial used in bone repair and regeneration fulfils all the requirements for an ideal bone graft. In this study hydroxyapatite/polycaprolactone (HA/PCL) composite scaffolds were prepared by a wet chemical method at room temperature. The physico-chemical properties of the composite materials were characterized by X-ray diffraction, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, while scaffold morphology was investigated by scanning electron microscopy (SEM) with energy-dispersive spectroscopy to validate the process used for synthesis. Finally, the response of bone marrow-derived human mesenchymal stem cells (hMSCs) in terms of cell proliferation and differentiation to the osteoblastic phenotype was evaluated using the Alamar blue assay, SEM and alkaline phosphatase activity. Microstructural analysis indicated that the HA particles were distributed homogeneously within the PCL matrix. The biological results revealed that the HA/PCL composite scaffolds are suitable for the proliferation and differentiation of MSCs in vitro, supporting osteogenesis after 15 days. All the results indicate that these scaffolds meet the requirements of materials for bone tissue engineering and could be used for many clinical applications in orthopaedic and maxillofacial surgery.

Mast cells have a protumorigenic role in human thyroid cancer.

In different human carcinoma types, mast cell infiltrate increases with respect to normal tissue and mast cell density correlates with a bad prognosis. To assess the role of mast cells in human thyroid cancer, we compared the density of tryptase-positive mast cells in 96 papillary thyroid carcinomas (PTCs) versus normal thyroid tissue from 14 healthy individuals. Mast cell density was higher in 95% of PTCs (n=91) than in control tissue. Mast cell infiltrate correlated with extrathyroidal extension (P=0.0005) of PTCs. We show that thyroid cancer cell-line-derived soluble factors induce mast cell activation and chemoattraction in vitro. Different mast cell lines (HMC-1 and LAD2) and primary human lung mast cells induced thyroid cancer cell invasive ability, survival and DNA synthesis in vitro. The latter effect was mainly mediated by three mast-cell-derived mediators: histamine, and chemokines CXCL1/GROα and CXCL10/IP10. We show that xenografts of thyroid carcinoma cells (8505-C) could recruit mast cells injected into the tail vein of mice. Co-injection of human mast cells accelerated the growth of thyroid cancer cell (8505-C) xenografts in athymic mice. This effect was mediated by increased tumor vascularization and proliferation, and was reverted by treating mice with sodium cromoglycate (Cromolyn), a specific mast cell inhibitor. In conclusion, our study data suggest that mast cells are recruited into thyroid carcinomas and promote proliferation, survival and invasive ability of cancer cells, thereby contributing to thyroid carcinoma growth and invasiveness.

The synergic effect of polylactide fiber and calcium phosphate particle reinforcement in poly epsilon-caprolactone-based composite scaffolds.

In this work, three-dimensional porous composite scaffolds, based on poly(epsilon-caprolactone) (PCL), were fabricated through the combination of a filament winding technique and a phase inversion/salt leaching process. Sodium chloride crystals were used as the porogen agent, and poly(lactic acid) (PLA) fibers and calcium phosphates as reinforcement. The aim of the current work is to assess the effective synergistic role of bioactive particles (i.e. alpha-tricalcium phosphates (alpha-TCP)) and PLA fibers on the morphology and mechanical response of the final scaffold. Morphological investigations performed on fiber-reinforced composite scaffolds with different PCL/alpha-TCP volume ratios (0%, 13%, 20% and 26%) show a high porosity degree (ca. 80%), pore interconnection and a homogeneous distribution of pores within the scaffold. More specifically, a bimodal pore size distribution was observed. This comprised microporosity (pores with radii ranging from 0.1 to 10 microm, which were strictly related to solvent extraction) and macroporosity (pores with radii from 10 to 300 microm, which were ascribable to the leaching of porogen elements). Static compressive tests showed that the effect of alpha-TCP on the mechanical response was to increase the elastic modulus up to a maximum value of 2.21+/-0.24 MPa, depending on the concentration of alpha-TCP added. This effect may be explained through the interaction of calcium-deficient hydroxyapatite crystals, formed as a consequence of a hydrolysis reaction of alpha-TCP, and the fiber-reinforced polymer matrix. The correct balance between chemical composition and spatial organization of reinforcement systems allows the attainment of an ideal compromise between mechanical response and bioactive potential, facilitating the development of composite scaffolds for bone tissue engineering applications.

OPN/CD44v6 overexpression in laryngeal dysplasia and correlation with clinical outcome.

Laryngeal dysplasia is a common clinical concern. Despite major advancements, a significant number of patients with this condition progress to invasive squamous cell carcinoma. Osteopontin (OPN) is a secreted glycoprotein, whose expression is markedly elevated in several types of cancers. We explored OPN as a candidate biomarker for laryngeal dysplasia. To this aim, we examined OPN expression in 82 cases of dysplasia and in hyperplastic and normal tissue samples. OPN expression was elevated in all severe dysplasia samples, but not hyperplastic samples, with respect to matched normal mucosa. OPN expression levels correlated positively with degree of dysplasia (P=0.0094) and negatively with disease-free survival (P<0.0001). OPN expression was paralleled by cell surface reactivity for CD44v6, an OPN functional receptor. CD44v6 expression correlated negatively with disease-free survival, as well (P=0.0007). Taken as a whole, our finding identify OPN and CD44v6 as predictive markers of recurrence or aggressiveness in laryngeal intraepithelial neoplasia, and overall, point out an important signalling complex in the evolution of laryngeal dysplasia.

Effects of S-adenosylmethionine (SAMe) on doxorubicin-induced cardiotoxicity in the rat.

One of the most important factors that determine the therapeutic limits of doxorubicin (DXR), an anthracycline antibiotic, appears in acute and late cardiotoxicity. The aim of our investigations was to evaluate the effects of S-adenosylmethionine (SAMe), an important precursor of the organic sulfated compounds and a fundamental agent in physiologic transmethylation, on DXR-induced cardiotoxicity. For that purpose, normotensive Wistar rats were treated with DXR associated with or without SAMe. Pretreatment with SAMe significantly reduced DXR-induced electrocardiographic and morphologic changes, lethality, and body weight. The protective effect of SAMe may partly be due to its scavenger ability against free radicals and superoxides. In addition to its action as a fundamental precursor of the sulfated compounds, the principal agent in physiological transmethylations, and an increase of glutathione (GSH) synthesis.