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The present work targets the development of collagen-based hydrogel precursors, functionalized with photo-crosslinkable methacrylamide moieties (COL-MA), for vascular tissue engineering (vTE) applications. The developed materials were physico-chemically characterized in terms of crosslinking kinetics, degree of modification/conversion, swelling behavior, mechanical properties and in vitro cytocompatibility. The collagen derivatives were benchmarked to methacrylamide-modified gelatin (GEL-MA), due to its proven track record in the field of tissue engineering. To the best of our knowledge, this is the first paper in its kind comparing these two methacrylated biopolymers for vTE applications. For both gelatin and collagen, two derivatives with varying degrees of substitutions (DS) were developed by altering the added amount of methacrylic anhydride (MeAnH). This led to photo-crosslinkable derivatives with a DS of 74 and 96% for collagen, and a DS of 73 and 99% for gelatin. The developed derivatives showed high gel fractions (i.e. 74% and 84%, for the gelatin derivatives; 87 and 83%, for the collagen derivatives) and an excellent crosslinking efficiency. Furthermore, the results indicated that the functionalization of collagen led to hydrogels with tunable mechanical properties (i.e. storage moduli of [4.8-9.4 kPa] for the developed COL-MAs versus [3.9-8.4 kPa] for the developed GEL-MAs) along with superior cell-biomaterial interactions when compared to GEL-MA. Moreover, the developed photo-crosslinkable collagens showed superior mechanical properties compared to extracted native collagen. Therefore, the developed photo-crosslinkable collagens demonstrate great potential as biomaterials for vTE applications., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A fast re-endothelialization, along with the inhibition of neointima hyperplasia, are crucial to reduce the failure of vascular bypass grafts. Implants modifications with molecules capable of speeding up the re-endothelialization process have been proposed over the last years. However, clinical trials of angiogenic factor delivery have been mostly disappointing, underscoring the need to investigate a wider array of angiogenic factors. In this work, a drug release system based on a type I collagen hydrogel has been proposed for the controlled release of Pleiotrophin (PTN), a cytokine known for its pro-angiogenetic effects. Heparin, in virtue of its ability to sequester, protect and release growth factors, has been used to better control the release of PTN. Performances of the PTN drug delivery system on endothelial (ECs) and smooth muscle cells (SMCs) have been investigated. Structural characterization (mechanical tests and immunofluorescent analyses of the collagen fibers) was performed on the gels to assess if heparin caused changes in their mechanical behavior. The release of PTN from the different gel formulations has been analyzed using a PTN-specific ELISA assay. Cell viability was evaluated with the Alamar Blue Cell Viability Assay on cells directly seeded on the gels (direct test) and on cells incubated with supernatant, containing the released PTN, obtained from the gels (indirect test). The effects of the different gels on the migration of both ECs and SMCs have been evaluated using a Transwell migration assay. Hemocompatibility of the gel has been assessed with a clotting/hemolysis test. Structural analyses showed that heparin did not change the structural behavior of the collagen gels. ELISA quantification demonstrated that heparin induced a constant release of PTN over time compared to other conditions. Both direct and indirect viability assays showed an increase in ECs viability while no effects were noted on SMCs. Cell migration results evidenced that the heparin/PTN-modified gels significantly increased ECs migration and decreased the SMCs one. Finally, heparin significantly increased the hemocompatibility of the collagen gels. In conclusion, the PTN-heparin-modified collagen here proposed can represent an added value for vascular medicine, able to ameliorate the biological performance, and integration of vascular grafts.

Vascular tissue engineering combines cells with scaffold materials in vitro aiming the development of physiologically relevant vascular models. For natural scaffolds such as collagen gels, where cells can be mixed with the material solution before gelation, cell seeding density is a key parameter that can affect extracellular matrix deposition and remodeling. Nonetheless, this parameter is often overlooked and densities sensitively lower than those of native tissues, are usually employed. Herein, the effect of seeding density on the maturation of tubular collagen gel-based scaffolds cellularized with smooth muscle cells is investigated. The compaction, the expression, and deposition of key vascular proteins and the resulting mechanical properties of the constructs are evaluated up to 1 week of maturation. Results show that increasing cell seeding density accelerates cell-mediated gel compaction, enhances elastin expression (more than sevenfold increase at the highest density, Day 7) and finally improves the overall mechanical properties of constructs. Of note, the tensile equilibrium elastic modulus, evaluated by stress-relaxation tests, reach values comparable to native arteries for the highest cell density, after a 7-day maturation. Altogether, these results show that higher cell seeding densities promote the rapid maturation of collagen gel-based vascular constructs toward structural and mechanical properties better mimicking native arteries., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One of the tightest bottlenecks in vascular tissue engineering (vTE) is the lack of strength and elasticity of engineered vascular wall models caused by limited elastic fiber deposition. In this study, flat and tubular collagen gel-based scaffolds were cellularised with vascular smooth muscle cells (SMCs) and supplemented with human plasma fibronectin (FN), a known master organizer of several extracellular matrix (ECM) fiber systems. The consequences of FN on construct maturation was investigated in terms of geometrical contraction, viscoelastic mechanical properties and deposition of core elastic fiber proteins. FN was retained in the constructs and promoted deposition of elastin by SMCs as well as of several proteins required for elastogenesis such as fibrillin-1, lysyl oxidase, fibulin-4 and latent TGF-β binding protein-4. Notably, gel contraction, tensile equilibrium elastic modulus and elasticity were strongly improved in tubular engineered tissues, approaching the behaviour of native arteries. In conclusion, this study demonstrates that FN exerts pivotal roles in directing SMC-mediated remodeling of scaffolds toward the production of a physiological-like, elastin-containing ECM with excellent mechanical properties. The developed FN-supplemented systems are promising for tissue engineering applications where the generation of mature elastic tissue is desired and represent valuable advanced in vitro models to investigate elastogenesis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The risk of bacterial colonization of abiotic surfaces of biomedical devices poses important challenges for the pharmaceutical and biomaterials science fields. In this scenario, antibacterial coatings have been developed, using a number of different molecules and materials. Among them, chitosan is a non-cytotoxic, biocompatible biopolymer with an inherent antimicrobial activity that has been already used in a wide variety of healthcare and industrial applications. Herein, chitosan-based antibacterial coatings are critically surveyed, with a special emphasis on their production methods, pharmaceutical and biomedical applications, along with their pros and cons, and finally highlighting the key challenges to be faced and future perspectives in this field., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The development of tubular engineered tissues is a challenging research area aiming to provide tissue substitutes but also in vitro models to test drugs, medical devices, and even to study physiological and pathological processes. In this work, the design, fabrication, and validation of an original cost-effective tubular multilayered-tissue culture system (TMCS) are reported. By exploiting cellularized collagen gel as scaffold, a simple moulding technique and an endothelialization step on a rotating system, TMCS allowed to easily prepare in 48 h, trilayered arterial wall models with finely organized cellular composition and to mature them for 2 weeks without any need of manipulation. Multilayered constructs incorporating different combinations of vascular cells are compared in terms of cell organization and viscoelastic mechanical properties demonstrating that cells always progressively aligned parallel to the longitudinal direction. Also, fibroblast compacted less the collagen matrix and appeared crucial in term of maturation/deposition of elastic extracellular matrix. Preliminary studies under shear stress stimulation upon connection with a flow bioreactor are successfully conducted without damaging the endothelial monolayer. Altogether, the TMCS herein developed, thanks to its versatility and multiple functionalities, holds great promise for vascular tissue engineering applications, but also for other tubular tissues such as trachea or oesophagus., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Electrospun PET (ePET) is a promising material for small caliber vascular graft applications owing to its tunable mechanical properties, biocompatibility, and nanofibrous structure that mimic the morphology of natural extracellular matrix. However, the inherent inertness of PET impairs the adhesion and proliferation of endothelial cells on the inner surface of ePET tubular grafts, hindering the formation of a functional endothelium. Gelatin coatings, owing to their ability to promote endothelialization, are a valuable approach to overcome the limitations of ePET. Herein, a novel process for the deposition of stable biomimetic coatings of gelatin on ePET tubular grafts is proposed. Electrospun PET was first aminated by plasma treatment and then coated with a gelatin hydrogel cross-linked in situ by a Michael-type addition reaction. Amination provided a superhydrophilic behavior to the ePET surface, allowing easy gelatin interpenetration along the wall thickness of the tubular structure, and the obtainment of thin coatings that maintained the morphology of ePET fibers. Gelatin coating was stable at long term in a physiological-like environment, noncytotoxic and promoted in vitro cell adhesion and proliferation. Noteworthy, the mechanical properties of gelatin-coated ePET tubular grafts were improved in terms of elastic modulus, compliance, and elastic recoil, finally better matching the characteristics of native blood vessels. Altogether, the proposed coating technique successfully combines the advantages of ePET nanofibrous structure with cross-linked gelatin biological cues and mechanical reinforcement, and emerges as a promising strategy for the development of biocompatible small caliber vascular grafts with superior biomimetic and mechanical properties. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2405-2415, 2017., (© 2017 Wiley Periodicals, Inc.)

Tissue engineering is a promising alternative to autografts or allografts for the regeneration of large bone defects. Cell-free biomaterials with different degrees of sophistication can be used for several therapeutic indications, to stimulate bone repair by the host tissue. However, when osteoprogenitors are not available in the damaged tissue, exogenous cells with an osteoblast differentiation potential must be provided. These cells should have the capacity to colonize the defect and to participate in the building of new bone tissue. To achieve this goal, cells must survive, remain in the defect site, eventually proliferate, and differentiate into mature osteoblasts. A critical issue for these engrafted cells is to be fed by oxygen and nutrients: the transient absence of a vascular network upon implantation is a major challenge for cells to survive in the site of implantation, and different strategies can be followed to promote cell survival under poor oxygen and nutrient supply and to promote rapid vascularization of the defect area. These strategies involve the use of scaffolds designed to create the appropriate micro-environment for cells to survive, proliferate, and differentiate in vitro and in vivo. Hydrogels are an eclectic class of materials that can be easily cellularized and provide effective, minimally invasive approaches to fill bone defects and favor bone tissue regeneration. Furthermore, by playing on their composition and processing, it is possible to obtain biocompatible systems with adequate chemical, biological, and mechanical properties. However, only a good combination of scaffold and cells, possibly with the aid of incorporated growth factors, can lead to successful results in bone regeneration. This review presents the strategies used to design cellularized hydrogel-based systems for bone regeneration, identifying the key parameters of the many different micro-environments created within hydrogels., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Although branched and linear polyethylenimines (bPEIs and lPEIs) are gold standard transfectants, a systematic analysis of the effects of the preparation protocol of polyplexes and the composition of the transfection medium on their physicochemical behaviour and effectiveness in vitro have been much neglected, undermining in some way the identification of precise structure-function relationships. This work aimed to address these issues. bPEI/DNA and lPEI/DNA, prepared using two different modes of addition of reagents, gave rise to polyplexes with exactly the same chemical composition but differing in dimensions. Upon dilution in serum-free medium, the size of any kind of polyplex promptly rose over time while remained invariably stable in complete DMEM. Of note, the bigger the dimension of polyplexes (in the nano- to micrometer range), the greater their efficiency in vitro. Besides, centrifugal sedimentation of polyplexes displaying different dimensions to speed up and enhance their settling onto cells boosted transfection efficiencies. Conversely, transgene expression was significantly blunted in cells held upside-down and transfected, definitively pointing out the impact of gravitational sedimentation of polyplexes on their transfection efficiency. Overall, much more attention must be paid to the actual polyplex size that relies on the complexation conditions and the transfection medium.

The acquisition of new thorough knowledge on the interactions existing between vascular cells would represent a step forward in the engineering of vascular tissues. In this light, herein we designed a physiological-like tri-culture in vitro vascular wall model using a planar cellularized collagen gel as the scaffold. The model can be obtained in 24 h and features multi-layered hierarchical organization composed of a fibroblast-containing adventitia-like layer, a media-like layer populated by smooth muscle cells and an intima-like endothelial cell monolayer. After 7 days of static culture, the compaction of the collagen matrix by the vascular cells was achieved, and the deposition of the vascular extracellular matrix components fibronectin, fibrillin-1 and tropoelastin was observed. The blood-compatible functionality of the endothelial cell monolayer was demonstrated by a blood clotting assay: after 7 days of maturation, clotting was prevented on the endothelialized constructs (more than 80% free hemoglobin maintained after 60 min of blood contact) but not at all on non-endothelialized ones (less than 20% free hemoglobin). In addition, western blotting results suggested that in the tri-culture model the loss of smooth muscle cell phenotype was delayed compared to what was observed in the mono-culture model, finally resulting in a behaviour more similar to the in vivo conditions. Overall, our findings indicate that this in vitro model has the potential to be used as an advanced system to examine vascular cell behavioural interactions, as well as for drug testing and the investigation of physiological and pathological processes.

A number of smart nonviral gene delivery vectors relying on bioresponsiveness have been introduced in the past few years to overcome the limits of the first generation of gene carriers. Among them, redox-sensitive lipidic and polymeric vectors exploit the presence of disulfide bonds in their structure to take advantage of the highly reductive intracellular milieu and to promote complex unpacking and nucleic acids release after cellular uptake (disulfide linker strategy). Glutathione (GSH) has been often identified as the leading actor in the intracellular reduction of bioreducible vectors but their actual mechanisms of action have been rarely investigated in depth and doubts about the real effectiveness of the disulfide linker strategy have been raised. Herein, we outline a simple protocol for the preparation and investigation of nano-sized reducible cationic liposomes, focusing on their thorough characterization and optimization as gene delivery vectors. In addition, we carefully describe the techniques and procedures necessary for the assessment of the bioreducibility of the vectors and to demonstrate that the GSH-mediated intracellular cleavage of disulfide bonds is a pivotal step in their transfection process. Liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and of the reducible cationic lipid SS14 are reported as a practical example but the proposed protocol can be easily shifted to other formulations of reducible lipids/liposomes and to reducible polymers.

Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency.

The development of multifunctional vectors for efficient and safe gene delivery is one of the major challenges for scientists working in the gene therapy field. In this context, we have designed a novel type of aminoglycoside-rich dendrimers with a defined structure based on polyamidoamine (PAMAM) in order to develop efficient, nontoxic gene delivery vehicles. Three different conjugates, i.e., PAMAM G4-neamine, -paromomycin, and -neomycin, were synthesized and characterized by nuclear magnetic resonance (NMR) and MALDI analysis. The conjugates were found to self-assemble electrostatically with plasmid DNA, and unlike neamine conjugate, each at its optimum showed increased gene delivery potency compared to PAMAM G4 dendrimer in three different cell lines, along with negligible cytotoxicity. These results all disclosed aminoglycosides as suitable functionalities for tailoring safe and efficient multifunctional gene delivery vectors.

Although redox-sensitive transfectants have been considered hitherto as the Holy Grail of gene delivery because of their ability to restrict the release of nucleic acids to intracellular compartments, the reasons for their sometimes lackluster performance do not seem likewise clear. To ascertain the possible influence of extracellular soluble thiols, exofacial protein sulfhydryls (EPTs) and glutathione (GSH) on the overall efficacy of bioreducible lipoplexes, we utilized a cationic gemini surfactant--SS14--in which the two single-chain amphiphiles are held together by a suitable redox-sensitive linkage. We herein draw a big picture whereby the interaction of bioreducible lipoplexes with cells and their internalization are tightly coupled events that ultimately do affect transfection. Specifically, we provide evidence that EPTs entail the reduction-triggered disruption of SS14 lipoplexes in plain Dulbecco's Modified Eagle Medium (DMEM), thereby resulting in a considerable waste (ca. 30%) of nucleic acids and low transgene expression. The DNA release from bioreducible lipoplexes can be blunted to ca. 16% by transfecting cells in complete medium and fully reverted by preincubating them for 1h before delivery in the same culture supernatant (i.e. preconditioning), thus significantly increasing transfection by ca. 3-fold and 10-fold, respectively. These results lead to the proposal of the protein corona as the central mediator in shielding SS14 bioreducible lipoplexes from the action of EPTs in the early phase of delivery and provide a smart solution as to how to increase their efficacy. Besides, we pinpoint associations between intracellular GSH levels and the extent of transfection. All these issues were unique to bioreducible lipoplexes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Efficient delivery of nucleic acids into cells is a promising technique to modulate cellular gene expression for therapeutic and research applications. Cationic lipid-based liposomes represent one of the most intensively studied and employed nonviral vectors. They are positively charged at physiological pH and spontaneously self-assemble with polyanionic nucleic acids forming nanoscaled complexes named lipoplexes. Here, we draft a simple protocol for the development, characterization, optimization, and screening of liposomal formulations for in vitro gene delivery. In particular, we report as a practical example a quick method to formulate and extrude nanometer-sized unilamellar cationic vesicles composed of DOTAP as cationic lipid and DOPE as zwitterionic helper lipid at 1:1 molar ratio. The physico-chemical characterization of liposomes and lipoplexes involves the measurement of mean diameter and overall surface charge using Dynamic Light Scattering (DLS) and Laser Doppler Microelectrophoresis. The outlined transfection procedure takes into account several experimental parameters affecting the in vitro performance of gene delivery systems, paying special attention to the charge ratio (CR). Gene delivery effectiveness is evaluated both in terms of transfection efficiency and cytotoxicity of the vector to find the optimal transfection conditions. Importantly, the proposed protocol can be easily shifted to different types of nonviral vectors.

Background: Among the different causes of orthopedic and dental implant failure, infection remains the most serious and devastating complication associated with biomaterial devices., Purpose: The aim of this study was to develop an innovative osteointegrative and antibacterial biomimetic coating on titanium and to perform a chemical-physical and in vitro biological characterization of the coating using the SAOS-2 cell line. We also studied the antibacterial properties of the coating against both Gram-positive and Gram-negative bacteria strains., Methods: An electrochemical solution containing silicon, calcium, phosphorous, sodium, and silver nanoparticles was used to obtain the antibacterial by Anodic Spark Deposition (ASD) treatment. Surface morphology was characterized using SEM and laser profilometry. A qualitative analysis of the chemical composition of the coating was assessed by EDS. The adhesion properties of the coating to the titanium bulk were performed with a 3-point bending test. SAOS-2 osteoblastic cell line spreading and morphology and viability were investigated. The bacterial adhesion and the antibacterial properties were investigated after 3 h and 24 h of incubation with Streptococcus mutans, Streptococcus epidermidis, and Escherichia coli bacterial strains., Results: The proposed anodization treatment created a chemically and morphologically modified, adherent titanium oxide layer, characterized by a microporous morphology enriched by calcium, silicon, phosphorous, and silver. The preliminary biological characterization showed optimal SAOS-2 cell adhesion and proliferation as well as a strong antibacterial effect., Conclusions: Based on the results of this study, we believe that this novel biomimetic and antibacterial treatment hold promise for enhancing osteointegration while conferring strong antibacterial properties to titanium.

Gene therapy is emerging as a revolutionary alternative to conventional therapeutic approaches. However, its clinical application is still hampered by the lack of safe and effective gene delivery techniques. Among the plethora of diverse approaches used to ferry nucleic acids into target cells, non-viral vectors represent promising and safer alternatives to viruses and physical techniques. Both cationic lipids and polymers spontaneously wrap and shrink the genetic material in complexes named lipoplexes and polyplexes, respectively, thereby protecting it and shielding its negative charges. The development of non-viral vectors commenced more than two decades ago. Since then, some major classes of interesting molecules have been identified and modified to optimize their properties. However, the way towards the final goal of gene delivery, i.e. protein expression or gene silencing, is filled with obstacles and current non-viral carriers still have concerns about their overall efficiency. We strongly believe that the future of non-viral gene delivery relies on the development of multifunctional vectors specifically tailored with diverse functionalities that act more like viruses. Although these vectors are still a long way from clinical practice they are the ideal platform to effectively shuttle the genetic material to target cells in a safe and controlled way. In this review, after briefly introducing the basis of gene delivery and therapeutic applications we discuss the main polymeric and lipidic vectors utilized for gene delivery, focusing on the strategies adopted to overcome the major weaknesses inherent to their still limited activity, on the way towards ideal multifunctional vectors.

Background: Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI(x)) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked., Methodology/principal Findings: With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI(x) derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI(x) series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI(x) copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI(2.7%) was as effective as and less cytotoxic than the gold standard 25 kDa bPEI., Conclusions/significance: This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI(x) copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity.

We present an integrated experimental-computational mechanobiology model of chondrogenesis. The response of human articular chondrocytes to culture medium perfusion, versus perfusion associated with cyclic pressurisation, versus non-perfused culture, was compared in a pellet culture model, and multiphysic computation was used to quantify oxygen transport and flow dynamics in the various culture conditions. At 2 weeks of culture, the measured cell metabolic activity and the matrix content in collagen type II and aggrecan were greatest in the perfused+pressurised pellets. The main effects of perfusion alone, relative to static controls, were to suppress collagen type I and GAG contents, which were greatest in the non-perfused pellets. All pellets showed a peripheral layer of proliferating cells, which was thickest in the perfused pellets, and most pellets showed internal gradients in cell density and matrix composition. In perfused pellets, the computed lowest oxygen concentration was 0.075 mM (7.5% tension), the maximal oxygen flux was 477.5 nmol/m(2)/s and the maximal fluid shear stress, acting on the pellet surface, was 1.8 mPa (0.018 dyn/cm(2)). In the non-perfused pellets, the lowest oxygen concentration was 0.003 mM (0.3% tension) and the maximal oxygen flux was 102.4 nmol/m(2)/s. A local correlation was observed, between the gradients in pellet properties obtained from histology, and the oxygen fields calculated with multiphysic simulation. Our results show up-regulation of hyaline matrix protein production by human chondrocytes in response to perfusion associated with cyclic pressurisation. These results could be favourably exploited in tissue engineering applications.

Background: A promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes). On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH), these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined., Methodology/principal Findings: With the aim of addressing this issue, SS14 was here formulated into binary and ternary 100 nm-extruded liposomes and the effects of the helper lipid composition and of the SS14/helper lipids molar ratio on chemical-physical and structural parameters defining transfection effectiveness were investigated. Among all formulations tested, DOPC/DOPE/SS14 at 25:50:25 molar ratio was the most effective in transfection studies owing to the presence of dioleoyl chains and phosphatidylethanolamine head groups in co-lipids. The increase in SS14 content up to 50% along DOPC/DOPE/SS14 liposome series yielded enhanced transfection, up to 2.7-fold higher than that of the benchmark Lipofectamine 2000, without altering cytotoxicity of the corresponding lipoplexes at charge ratio 5. Secondly, we specifically investigated the redox-dependent mechanisms of gene delivery into cells through tailored protocols of transfection in GSH-depleted and repleted vs. increased oxidative stress conditions. Importantly, GSH specifically induced DNA release in batch and in vitro., Conclusions/significance: The presence of helper lipids carrying unsaturated dioleoyl chains and phosphatidylethanolamine head groups significantly improved transfection efficiencies of DOPC/DOPE/SS14 lipoplexes. Most importantly, this study shows that intracellular GSH levels linearly correlated with transfection efficiency while oxidative stress levels did not, highlighting for the first time the pivotal role of GSH rather than oxidative stress in its whole in transfection of bioreducible vectors.

Background: Despite the use of currently optimized lipofection conditions, including transfection in serum-depleted media, the efficiency of gene transfer is low and high transfection rates often induce cytotoxicity. A lipid formulation with transfection efficiency not inhibited by serum would provide an advance towards in vivo applications., Methods: We explored the ability of the cationic lipid SH-14 to dimerize upon DNA and form lipoplexes, and potentially release nucleic acids in the intracellular reducing milieu. We investigated the critical micelle-forming concentration of SH-14 and its intrinsic toxicity, size and Zeta potential measurements, the in vitro cytotoxicity of SH-14/plasmid DNA lipoplexes and their ability to transfect cells., Results: Among all the charge ratios (CR, + /-) tested, lipoplexes at CR 10 with a mean diameter of 295 nm and a surface charge of + 20 mV, exhibited the best compromise between transfection efficiency and tolerability. SH-14 presented the same cytotoxicity level whether alone or complexed in lipoplexes. Lipofections carried out in serum-free medium shared a transfection efficiency, on average, of 40% and a cytotoxicity of 38%. An increase of 73% in transfection efficiency and 24% in cell viability were obtained, extending lipofection over 48 h in complete-medium. Moreover, when serum concentration was increased from 10% to 50%, a three-fold increase in plasmid dose led to more than 72% of cells being transfected with almost no sign of cytotoxicity., Conclusions: Overall, SH-14 presents good potential as a novel transfection reagent to be used in the presence of serum.

An auxological and endocrinological study was performed in 21 thalassaemic patients with growth retardation and skeletal dysplasia secondary to desferrioxamine. Bone metaphyseal proximal tibial or iliac crest biopsy was performed in six patients with severe genu valgum or non-traumatic vertebral compression. GH insufficiency/deficiency (GH deficiency: peak after stimulation test below 6 ng/ml) was found in 72% of our thalassaemic patients with skeletal dysplasia, but in only 41% of patients without skeletal dysplasia. Bone histology showed abnormal chondrocytes, alteration of staining pattern of cartilage, irregular columnar cartilage and lacunae in the cartilaginous tissue. The behaviour of bone tissue was unpredictable (presence of thick or thin osteoid layer). Bone microfractures were sometimes present. The bone microstructure showed scarce mineralization, which was evenly or irregularly distributed. The bone tissue apatitic phase was quantitatively reduced. The hardness of bone tissue was remarkably lower than that of normal bone in three out of six patients. In conclusion, iron chelation therapy in patients with acquired skeletal dysplasia seems to interfere with GH secretion. The early identification of clinical and radiological abnormalities of skeletal dysplasia is of paramount importance in preventing severe bone destruction.