Adipose-derived stem cells could sense the nano-scale cues as myogenic-differentiating factors.

Microenvironmental cues, such as surface topography and substrate stiffness, may affect stem cells adhesion, morphology, alignment, proliferation and differentiation. Adipose derived stem cells (ASCs) have attracted considerable interest in regenerative medicine due to their easy isolation, extensive in vitro expandability and ability to differentiate along a number of different tissue-specific lineages. The aim of this work was to investigate ASCs adhesion, alignment and differentiation into myogenic lineage on nanofibrous polymeric scaffolds with anisotropic topography. Nanostructured scaffolds with randomized or parallel fibers were fabricated by electrospinning using polycaprolactone (PCL) and the polycarbonate-urethane ChronoFlex AL 80A (CFAL). Cells expressed myosin (fast skeletal) and tropomyosin in all surface topographies 7 days after seeding but myotube formation was only observed on CFAL scaffolds and only few myotubes were formed on PCL scaffolds. The different cell behavior could be ascribed to two main parameters: fibers dimensions and fibers orientation of the substrates that could result in a better myotube formation on CFAL scaffolds.

Pectin-based injectable biomaterials for bone tissue engineering.

A variety of natural polymers and proteins are considered to be 3D cell culture structures able to mimic the extracellular matrix (ECM) to promote bone tissue regeneration. Pectin, a natural polysaccharide extracted from the plant cell walls and having a chemical structure similar to alginate, provides interesting properties as artificial ECM. In this work, for the first time, pectin, modified with an RGD-containing oligopeptide or not, is used as an ECM alternative to immobilize cells for bone tissue regeneration. The viability, metabolic activity, morphology, and osteogenic differentiation of immobilized MC3T3-E1 preosteoblats demonstrate the potential of this polysaccharide to keep immobilized cells viable and differentiating. Preosteoblasts immobilized in both types of pectin microspheres maintained a constant viability up to 29 days and were able to differentiate. The grafting of the RGD peptide on pectin backbone induced improved cell adhesion and proliferation within the microspheres. Furthermore, not only did cells grow inside but also they were able to spread out from the microspheres and to organize themselves in 3D structures producing a mineralized extracellular matrix. These promising results suggest that pectin can be proposed as an injectable cell vehicle for bone tissue regeneration.

Structural properties of polysaccharide-based microcapsules for soft tissue regeneration.

Autologous and eterologous cell encapsulation has been extensively studied for clinical application in functional organs substitution, recombinant cell transplantation in gene therapy or in muscle and cartilage regeneration to treat degenerative pathologies. In this work, calcium alginate, calcium alginate/chitosan, calcium alginate/gelatin and pectin/chitosan microcapsules were prepared to be used as innovative injectable scaffolds for soft issue regeneration by a simple extrusion method from aqueous solutions. Prepared microcapsules had spherical morphology, whereas their size was deeply influenced by the polymeric composition. When incubated in a physiological-like environment up to 30 days, they underwent an initial swelling, followed by weight loss at different rates, depending on the microcapsules formulation. The encapsulation of mouse myoblast cells (C2C12 cell line) was obtained in calcium alginate, calcium alginate/chitosan, calcium alginate/gelatin microcapsules. Cells were alive throughout the encapsulation procedure, and were recovered by a mechanical rupture of the microcapsules. After 7 days, fractured microcapsules led cells to migrate gradually out.

Ability of polyurethane foams to support placenta-derived cell adhesion and osteogenic differentiation: preliminary results.

In bone tissue reconstruction, the use of engineered constructs created by mesenchymal stem cells (MSCs) that differentiate and proliferate into 3D porous scaffolds is an appealing alternative to clinical therapies. Human placenta represents a possible source of MSCs, as it is readily available without invasive procedures and because of the phenotypic plasticity of many of the cell types isolated from this tissue. The scaffold considered in this work is a slowly degradable polyurethane foam (EF PU foam), synthesized and characterized for morphology and in vitro interaction with chorion mesenchymal cells (CMCs). These cells were isolated from human term placenta and cultured onto the EF PU foam using two different culture media (EMEM and NH osteogenic differentiation medium). Synthesized EF PU foam showed homogeneous pore size and distribution, with 89% open porosity. In vitro tests showed CMCs scaffold colonization, as confirmed by Scanning Electron Microscopy (SEM) observations and hematoxylin-eosin staining. Alizarin Red staining revealed the presence of a small amount of calcium deposition for the samples treated with the osteogenic differentiation medium. Therefore, the proposed EF PU foam appears to stimulate cell adhesion in vitro, sustaining CMCs growth and differentiation into the osteogenic lineage.

Polyurethane foam/nano hydroxyapatite composite as a suitable scaffold for bone tissue regeneration.

In bone tissue regeneration, the use of biomineralized scaffolds to create the 3D porous structure needed for well-fitting with defect size and appropriate cell interactions, is a promising alternative to autologous and heterologous bone grafts. Biomineralized polyurethane (PU) foams are here investigated as scaffold for bone tissue regeneration. Biomineralization of the foams was carried out by activation of PU surface by a two steps procedure performed for different times (1 to 4 weeks). Scaffolds were investigated for morphological, chemico-physical and mechanical properties, as well as for in vitro interaction with rat Bone Marrow Mesenchymal Stem Cells (BMSCs). Untreated and biomineralized PU samples showed a homogenous morphology and regular pore size (average Ø=407µm). Phase and structure of formed calcium phosphates (CaPs) layer onto the PU foam were analyzed by Fourier Transform Infrared spectroscopy and X-ray diffraction, proving the formation of bone-like nano hydroxyapatite. Biomineralization caused a significant increase of mechanical properties of treated foams compared to untreated ones. Biomineralization also affected the PU scaffold cytocompatibility providing a more appropriate surface for cell attachment and proliferation. Considering the obtained results, the proposed scaffold can be considered suitable for bone tissue regeneration.

Bioabsorbable scaffold for in situ bone regeneration.

A non-porous poly-DL-lactide tubular chamber filled by demineralised bone matrix (DBM) and bone marrow stromal cells (BMSC) in combination, was evaluated as a scaffold for guided bone regeneration (GBR) in an experimental model using the rabbit radius. The tubular chamber had an internal diameter of 4.7 mm, a wall thickness of 0.4 mm and a length of 18 mm. Autologous BMSC were obtained, under general anaesthesia from rabbit iliac crest and isolated by centrifugation technique. Allogenic DBM was obtained from cortico-cancellous bone of rabbits. In general anaesthesia, a 10-mm defect was bilaterally created in the radii of 10 rabbits. On the right side (experimental side) the defect was bridged with the chamber filled with both BMSC and DBM. On the left side (control side) the defect was treated by positioning DBM and BMSC between the two stumps. At an experimental time of 4 months histology and histomorphometry demonstrated that the presence of a tubular chamber significantly improved bone regrowth in the defect The mean thickness of newly-formed bone inside the chamber was about 56.7+/-3.74% of the normal radial cortex, in comparison with 46.7+/-10.7% when DBM and BMSC without the chamber were placed in the defect, P<0.05). These results confirmed the effectiveness of the chamber as a container for factors promoting bone regeneration.

Antibacterial activity of zinc modified titanium oxide surface.

Titanium-based implants are successfully used for various biomedical applications. However, in some cases, e.g. in dental implants, failures due to bacterial colonization are reported. Surface modification is a commonly proposed strategy to prevent infections. In this work, titanium oxide, naturally occurring on the surface of titanium, was modified by promoting the formation of a mixed titanium and zinc oxide, on the basis of the idea that zinc oxide on titanium surface may act as the zinc oxide used in pharmaceutical formulation for its lenitive and antibacterial effects. The present work shows that it is possible to form a mixed titanium and zinc oxide on titanium surfaces, as shown by Scanning Electron Microscopy and XPS analysis. To this end titanium was preactivated by UV on crystalline titanium oxide, both in the anatase form or in the co-presence of anatase and rutile. By performing antibacterial assays, we provide evidence of a significant reduction in the viability of five streptococcal oral strains on titanium oxide surfaces modified with zinc. In conclusion, this type of chemical modification of titanium oxide surfaces with zinc might be considered a new way to reduce the risk of bacterial colonization, increasing the lifetime of dental system applications.

Calcified matrix production by SAOS-2 cells inside a polyurethane porous scaffold, using a perfusion bioreactor.

The repair and regeneration of damaged or resected bone are problematic. Bone autografts show optimal skeletal incorporation, but often bring about complications. Hence, there is increasing interest in designing new biomaterials that could potentially be used in the form of scaffolds as bone substitutes. In this study we used a hydrophobic cross-linked polyurethane in a typical tissue-engineering approach, that is, the seeding and in vitro culturing of cells within a porous scaffold. The polyurethane porous scaffold had an average pore diameter of 624 microm. Using a perfusion bioreactor, we investigated the effect of shear stress on SAOS-2 human osteoblast proliferation and calcified matrix production. The physical, morphological, and compressive properties of the polyurethane foam were characterized. At a scaffold perfusion rate of 3 mL/min, in comparison with static conditions without perfusion, we observed 33% higher cell proliferation; higher secretion of osteopontin, osteocalcin, decorin, and type I collagen (9.16-fold, 71.9-fold, 30.6-fold, and 18.12-fold, respectively); and 10-fold increased calcium deposition. The design of the bioreactor and the design of the polyurethane foam aimed at obtaining cell colonization and calcified matrix deposition. This cultured biomaterial could be used, in clinical applications, as an osteoinductive implant for bone repair.

Programmed cell delivery from biodegradable microcapsules for tissue repair.

Injectable and resorbable hydrogels are an extremely attractive class of biomaterials. They make it possible to fill tissue defects accurately with an undoubtedly minimally invasive approach and to locally deliver cells that support repair or regeneration processes. However, their use as a cell carrier is often hindered by inadequate diffusion in bulk. A possible strategy for overcoming this transport limitation might be represented by injection of rapidly degradable cell-loaded microcapsules, so that maximum material thickness is limited by sphere radius. Here, the possibility of achieving programmable release of viable cells from alginate-based microcapsules was explored in vitro, by evaluating variations in material stability resulting from changes in hydrogel composition and assessing cell viability after encapsulation and in vitro release from microcapsules. Degradation of pure alginate microspheres was varied from a few days to several weeks by varying sodium alginate and calcium chloride concentrations. The addition of poloxamer was also found to accelerate degradation significantly, with capsule breakdown almost complete by two weeks, while chitosan was confirmed to strengthen alginate cross-linking. The presence of viable cells inside microspheres was revealed after encapsulation, and released cells were observed for all the formulations tested after a time interval dependent on bead degradation speed. These findings suggest that it may be possible to fine tune capsule breakdown by means of simple changes in material formulation and regulate, and eventually optimize, cell release for tissue repair.

Micro- and nano-hydroxyapatite as active reinforcement for soft biocomposites.

Pectin-based biocomposite hydrogels were produced by internal gelation, using different hydroxyapatite (HA) powders from commercial source or synthesized by the wet chemical method. HA possesses the double functionality of cross-linking agent and inorganic reinforcement. The mineralogical composition, grain size, specific surface area and microstructure of the hydroxyapatite powders are shown to strongly influence the properties of the biocomposites. Specifically, the grain size and specific surface area of the HA powders are strictly correlated to the gelling time and rheological properties of the hydrogels at room temperature. Pectin pH is also significant for the formation of ionic cross-links and therefore for the hydrogels stability at higher temperatures. The obtained results point out that micrometric-size hydroxyapatite can be proposed for applications which require rapid gelling kinetics and improved mechanical properties; conversely the nanometric hydroxyapatite synthesized in the present work seems the best choice to obtain homogeneous hydrogels with more easily controlled gelling kinetics.

Synthesis and pharmacological evaluation of poly(oxyethylene) derivatives of 4-isobutylphenyl-2-propionic acid (ibuprofen).

The synthesis of three oligomeric derivatives of 4-isobutylphenyl-2-propionic acid (ibuprofen), namely, the monoester of tetraethylene glycol (I) and the diesters of poly(oxyethylene) samples having molecular weights of 1000 (+/- 50) and 2000 (+/- 150) (II and III), has been performed via the imidazolide method. The antiinflammatory activity of I-III, and of equivalent amounts of free drug, was determined in the carrageenan-induced rat paw edema assay at different times after oral administration and found to be considerably prolonged in the case of the three derivatives. The lowest molecular weight derivative (I) also had an enhanced initial activity with regard to 4-isobutylphenyl-2-propionic acid. These results were confirmed by measuring the plasma levels of 4-isobutylphenyl-2-propionic acid in rats at different times after oral administration.

New polymeric and oligomeric matrices as drug carriers.

The preparation of polymeric derivatives of drugs, in which drug moieties are covalently linked to polymeric or oligomeric matrices, in such a way that they can be released at the site of action, is one of the most promising ways to achieve results which often can be hardly obtained by other means, such as, for instance, better adsorption by some ways of administration (e.g., oral administration), preferential localization in the body, and longer duration of activity. The aim of this review is to provide an up-to-date picture of the state of art in this field. The synthetic aspects of the preparation of polymeric derivatives of drugs will be discussed, with special emphasis on general methods. The main criteria for selecting a particular type of matrix, and a particular bond between drug and matrix, in order to achieve a given purpose, will be also discussed. The main pharmacological results so far obtained by this technique will be emphasized.

Synthesis and characterization of poly(amido-amine)s belonging to two different homologous series.

Systematic work on the synthesis and characterization of two series (referred to as type A and B) of poly(amido-amine)s with tertiary amino-groups in the main chain is reported. The polymers were synthesized by polyaddition of alpha, omega-bis-(methyl-amino) alkanes with 1,4-bis-acryloylpiperazine (type A) and 1,12-bis-acryloyl-n-diaminododecane (type B). These materials, which will be employed in the preparation of block and graft copolymers for biomedical use, have been characterized by means of various techniques and the most interesting features are reported.

Grafting reactions and heparin adsorption of poly(amidoamine)-grafted poly(urethane amide)s.

Differently terminated poly(amidoamine) (PAA) oligomers were grafted on the surface of poly(ether urethane amide)s (PEUAm), with fumaric or maleic acid moieties. The grafting reaction was Michael-type addition of amino groups to activated double bonds in the PEUAm backbone. PAAs having primary amino, or secondary amino end-groups were directly grafted on the surface of PEUAm sheets. For vinyl-terminated chains an alpha, omega amino-polyether spacer was introduced initially, following the same addition mechanism. Ungrafted and grafted materials were characterized, besides other analytical techniques, by ATR FT-IR spectroscopy. The heparin adsorption on PEAUm films was analysed after its elution from heparinized samples, quantified by coagulation tests (aPTT), and related to the presence of the PAAs chains grafted on to the surface. Results indicate that PAA-grafted PEUAm elastomeric biomaterials, display enhanced heparin adsorption abilities.

Heparinizable graft copolymers from chlorosulphonated polyethylene with poly(amido-amine) segments.

The synthesis and the physical characterization of three graft copolymers (PES/PAA) obtained from chlorosulphonated polyethylene (PECS) and three different secondary amino end-capped poly(amido-amine)s are reported. The properties of these heparinizable materials appear to be suitable for constructing prosthetic devices for biomedical use. The heparin adsorbing ability and the stability of the complex with heparin of the three copolymers have been evaluated, by means of biological tests, as activated Partial Thromboplastin Time (PTT) and Thrombin Time (TT).

Heparin adsorbing capacities at physiological pH of three poly(amido-amine) resins, and of poly(amido-amine)-surface-grafted glass microspheres.

Three poly(amido-amine)s of similar structure in the form of highly hydrophilic crosslinked resins, have been prepared, and tested for their heparin-adsorbing capacity at physiological pH. They showed different capacities, and their capacities were related to their basicities. One of the same polymers was grafted on the surface of glass microspheres. After treatment, it was shown that the microspheres could adsorb significant amounts of heparin. In all cases most of the adsorbed heparin was hardly eluted with saline, plasma, or blood, but could be recovered by eluting with 0.1 M NaOH. The resins were found to have some haemolytic properties, but no haemolysis was observed with the grafted microspheres.

Genotoxicity of N-acryloyl-N'-phenylpiperazine, a redox activator for acrylic resin polymerization.

N-Acryloyl-N'-phenylpiperazine is a promoter of redox reactions synthesized recently, and proposed as an activator for the polymerization of acrylic resins for biomedical use. The chemical was analyzed for different genotoxicity endpoints, to obtain both information on its possible mutagenic/carcinogenic potential and a model analysis of a tertiary arylamine, which belongs to a class of chemicals commonly used as polymerization accelerators in the biomaterial field. The genotoxicity endpoints considered were: gene mutation in the Salmonella test; structural and numerical chromosome alterations in Chinese hamster V79 cells, evaluated by the micronucleus test together with an immunofluorescent staining specific for kinetochore proteins; in vitro and in vivo DNA damage, evaluated in V79 cells and in mouse liver by the alkaline DNA elution technique. On the whole, the results indicate that N-acryloyl-N'-phenylpiperazine is to be regarded not so much as a DNA-damaging agent, but as a genomic mutagen. Indeed, it was not mutagenic in Salmonella (though its toxicity did not allow testing concentrations over 70 micrograms/plate), and it was weakly positive in inducing chromosomal fragmentation in vitro (one positive, not dose-related, result out of five different doses tested) and in vivo DNA damage (increases in DNA elution rate never doubling control values). The chemical was, however, clearly positive (with dose-dependent effects up to about 25 times the control value) in causing numerical chromosome alterations, at the maximal non-toxic doses.

Polyurethane-coated, self-expandable biliary stent: an experimental study.

RATIONALE AND OBJECTIVES: We describe a self-expanding metallic biliary Gianturco-Rösch stent coated with polymeric material. The coating was designed to prevent the growth of neoplastic and reactive tissue within the biliary ducts. METHODS: The stents were coated with a solvent-casting technique, which consists of dissolving polyurethane (polyether urethane or polycarbonate urethane) pellets in a solvent (dimethylacetamide), dipping the stent in the solution, and completely evaporating the solvent. In vitro mechanical characterization of the stent was performed to determine the adhesion of the coating to the metallic cage, the best introducer caliber for implantation of the device, and the relationship between the stent's diameter and radial stress. RESULTS: Reports in the literature on the biostability of polycarbonate urethane compared with polyether urethane prompted us to use the former material to coat the stents. The solvent technique gives a smooth internal surface of the stent wall, leaving in relief the coated structure of the stent on the external surface. The functional tests demonstrated that the coating did not compromise the original characteristics of the stent in terms of self-expandability, axial flexibility, and increased radial rigidity of the device. CONCLUSION: Functional tests verified coating stability and device handling, which are the first steps toward in vivo experimentation.

In vitro stability of polyether and polycarbonate urethanes.

The in vitro structural stability of poly-ether-urethanes (PEUs) and poly-carbonate-urethanes (PCUs) was examined under strong acidic (HNO3) or alkaline (NaClO) oxidative conditions and in presence of a constant strain state. Polyurethane (PU) samples were represented by sheets solvent-cast from commercial pellets or by tubular specimens cut from commercial catheters. The specimens were strained at 100% uniaxial elongation over appropriate extension devices and completely immersed into the oxidative solutions at 50 degrees C for 7-14 days. The changes induced by the oxidative treatments were then evaluated by molecular weight analysis, tensile mechanical tests, and scanning electron microscopy. In the experiments with solvent-cast samples, the PEU Pellethane was degraded more in the alkaline oxidative conditions and mainly in the absence of an applied uniaxial stress. All the tested PCUs were, on the contrary, more affected by the acidic oxidative agent. All the PCUs proved to have overall better stability than the PEU. The susceptibility to oxidation was also dependent on the shape and bulk/surface organisation acquired by the same polymer during its processing. When the oxidative test was applied to catheters made of a PEU and a PCU, the results confirmed the better stability of poly-carbonate-urethanes.

[Biodegradation of dacron vascular prostheses. Physico-chemical, histological, morphometric and ultrastructural study]. / Biodegradazione delle protesi vascolari in Dacron. Studio chimico-fisico, istologico, morfometrico ed ultrastrutturale.

The paper deals with study of long-term stability as far as concerns Dacron vascular prostheses in woven and knitted double velour. Among our vascular prostheses case-reports, we evaluated three of them explanted after 11, 12, 20 years; all of the prostheses were patent. Chemical-physical, histopathological and ultrastructural analysis have been carried on in order to evaluate in vivo ageing of the examined prostheses. The results all indicate strong alterations of the original properties related to double velour of knitted prostheses and weak alterations of woven one.