Levofloxacin loaded poly (ethylene oxide)-chitosan/quercetin loaded poly (D,L-lactide-co-glycolide) core-shell electrospun nanofibers for burn wound healing.

This study developed a new burn wound dressing based on core-shell nanofibers that co-deliver antibiotic and antioxidant drugs. For this purpose, poly(ethylene oxide) (PEO)-chitosan (CS)/poly(D,L-lactide-co-glycolide) (PLGA) core-shell nanofibers were fabricated through co-axial electrospinning technique. Antibiotic levofloxacin (LEV) and antioxidant quercetin (QS) were incorporated into the core and shell parts of PEO-CS/PLGA nanofibers, respectively. The drugs could bond to the polymer chains through hydrogen bonding, leading to their steady release for 168 h. An in vitro drug release study showed a burst effect followed by sustained release of LEV and QS from the nanofibers due to the Fickian diffusion. The NIH 3T3 fibroblast cell viability of the drug loaded core-shell nanofibers was comparable to that in the control (tissue culture polystyrene) implying biocompatibility of the nanofibers and their cell supportive role. However, there was no significant difference in cell viability between the drug loaded and drug free core-shell nanofibers. According to in vivo experiments, PEO-CS-LEV/PLGA-QS core-shell nanofibers could accelerate the healing process of a burn wound compared to a sterile gauze. Thanks to the synergistic therapeutic effect of LEV and QS, a significantly higher wound closure rate was recorded for the drug loaded core-shell nanofibrous dressing than the drug free nanofibers and control. Conclusively, PEO-CS-LEV/PLGA-QS core-shell nanofibers were shown to be a promising wound healing material that could drive the healing cascade through local co-delivery of LEV and QS to burn wounds.

Propionate reinforces epithelial identity and reduces aggressiveness of lung carcinoma.

The epithelial-to-mesenchymal transition (EMT) plays a central role in the development of cancer metastasis and resistance to chemotherapy. However, its pharmacological treatment remains challenging. Here, we used an EMT-focused integrative functional genomic approach and identified an inverse association between short-chain fatty acids (propionate and butanoate) and EMT in non-small cell lung cancer (NSCLC) patients. Remarkably, treatment with propionate in vitro reinforced the epithelial transcriptional program promoting cell-to-cell contact and cell adhesion, while reducing the aggressive and chemo-resistant EMT phenotype in lung cancer cell lines. Propionate treatment also decreased the metastatic potential and limited lymph node spread in both nude mice and a genetic NSCLC mouse model. Further analysis revealed that chromatin remodeling through H3K27 acetylation (mediated by p300) is the mechanism underlying the shift toward an epithelial state upon propionate treatment. The results suggest that propionate administration has therapeutic potential in reducing NSCLC aggressiveness and warrants further clinical testing.

Topical systems for the controlled release of antineoplastic Drugs: Oxidized Alginate-Gelatin Hydrogel/Unilamellar vesicles.

The efficacy of chemotherapeutic procedures relies on delivering proper concentrations of anti-cancer drugs in the tumor surroundings, so as to prevent potential side effects on healthy tissues. Novel drug carrier platforms should not just be able to deliver anticancer molecules, but also allow for adjustements in the way these drugs are administered to the patients. We developed a system for delivering water-insoluble drugs, based on the use of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or bis(2-ethylhexyl) sulfosuccinate benzyl-n-hexadecyldimethylammonium (BHD-AOT), embedded into oxidized alginate-gelatin (ADA/Gel) hydrogel, emulating a patch for topic applications. After being loaded with curcumin, cancer cells such as human colorectal adenocarcinoma (HCT116 and DLD-1) and melanoma cell lines (MEL501), and non-malignant cells such as mammary epithelial cell lines (NMuMG) and embryonal fibroblasts (NIH 3T3 or NEO cells) were analyzed for biocompatibility and cytotoxic effects. The results show that the proposed system can load comparatively higher concentrations of the drug (with respect to other nano/microcarriers in the literature), and that it can enhance the likelihood of the drug being uptaken by cancer cells instead of non-malignant cells. These assays were complemented by diffusion studies across the stratum corneum of rat skin, with the aim of determining the system's efficiency during topical application. Finally, the stability of the patch was tested after lyophilization to determine its potential pharmaceutical use. As a whole, the combined system represents a highly reliable and robust method for embedding and delivering complex insoluble chemotherapeutical molecules, and it is less invasive than other alternative methods in the literature.

3D hydrogel-based microcapsules as an in vitro model to study tumorigenicity, cell migration and drug resistance.

In this work, we analyzed the reliability of alginate-gelatin microcapsules as artificial tumor model. These tumor-like scaffolds are characterized by their composition and stiffness (∼25 kPa), and their capability to restrict -but not hinder- cell migration, proliferation and release from confinement. Hydrogel-based microcapsules were initially utilized to detect differences in mechano-sensitivity between MCF7 and MDA-MB-231 breast cancer cells, and the endothelial cell line EA.hy926. Additionally, we used RNA-seq and transcriptomic methods to determine how the culture strategy (i.e. 2D v/s 3D) may pre-set the expression of genes involved in multidrug resistance, being then validated by performing cytotoxicological tests and assays of cell morphology. Our results show that both breast cancer cells can generate elongated multicellular spheroids inside the microcapsules, prior being released (mimicking intravasation stages), a behavior which was not observed in endothelial cells. Further, we demonstrate that cells isolated from 3D scaffolds show resistance to cisplatin, a process which seems to be strongly influenced by mechanical stress, instead of hypoxia. We finally discuss the role played by aneuploidy in malignancy and resistance to anticancer drugs, based on the increased number of polynucleated cells found within these microcapsules. Overall, our outcomes demonstrate that alginate-gelatin microcapsules represent a simple, yet very accurate tumor-like model, enabling us to mimic the most relevant malignant hints described in vivo, suggesting that confinement and mechanical stress need to be considered when studying pathogenicity and drug resistance of cancer cells in vitro. STATEMENT OF SIGNIFICANCE: In this work, we analyzed the reliability of alginate-gelatin microcapsules as an artificial tumor model. These scaffolds are characterized by their composition, elastic properties, and their ability to restrict cell migration, proliferation, and release from confinement. Our results demonstrate four novel outcomes: (i) studying cell migration and proliferation in 3D enabled discrimination between malignant and non-pathogenic cells, (ii) studying the cell morphology of cancer aggregates entrapped in alginate-gelatin microcapsules enabled determination of malignancy degree in vitro, (iii) determination that confinement and mechanical stress, instead of hypoxia, are required to generate clones resistant to anticancer drugs (i.e. cisplatin), and (iv) evidence that resistance to anticancer drugs could be due to the presence of polynucleated cells localized inside polymer-based artificial tumors.

Integration of Mesoporous Bioactive Glass Nanoparticles and Curcumin into PHBV Microspheres as Biocompatible Composite for Drug Delivery Applications.

Bioactive glasses (BGs) are being increasingly considered for biomedical applications. One convenient approach to utilize BGs in tissue engineering and drug delivery involves their combination with organic biomaterials in order to form composites with enhanced biocompatibility and biodegradability. In this work, mesoporous bioactive glass nanoparticles (MBGN) have been merged with polyhydroxyalkanoate microspheres with the purpose to develop drug carriers. The composite carriers (microspheres) were loaded with curcumin as a model drug. The toxicity and delivery rate of composite microspheres were tested in vitro, reaching a curcumin loading efficiency of over 90% and an improving of biocompatibility of different concentrations of MBGN due to its administrations through the composite. The composite microspheres were tested in terms of controlled release, biocompatibility and bioactivity. Our results demonstrate that the composite microspheres can be potentially used in biomedicine due to their dual effects: bioactivity (due to the presence of MBGN) and curcumin release capability.

3D Spheroids Versus 3D Tumor-Like Microcapsules: Confinement and Mechanical Stress May Lead to the Expression of Malignant Responses in Cancer Cells.

As 2D surfaces fail to resemble the tumoral milieu, current discussions are focused on which 3D cell culture strategy may better lead the cells to express in vitro most of the malignant hints described in vivo. In this study, this question is assessed by analyzing the full genetic profile of MCF7 cells cultured either as 3D spheroids-considered as "gold standard" for in vitro cancer research- or immobilized in 3D tumor-like microcapsules, by RNA-Seq and transcriptomic methods, allowing to discriminate at big-data scale, which in vitro strategy can better resemble most of the malignant features described in neoplastic diseases. The results clearly show that mechanical stress, rather than 3D morphology only, stimulates most of the biological processes involved in cancer pathogenicity, such as cytoskeletal organization, migration, and stemness. Furthermore, cells entrapped in hydrogel-based scaffolds are likely expressing other physiological hints described in malignancy, such as the upregulated expression of metalloproteinases or the resistance to anticancer drugs, among others. According to the knowledge, this study represents the first attempt to answer which 3D experimental system can better mimic the neoplastic architecture in vitro, emphasizing the relevance of confinement in cancer pathogenicity, which can be easily achieved by using hydrogel-based matrices.

Re-engineering Artificial Neoplastic Milieus: Taking Lessons from Mechano- and Topobiology.

Traditionally, cancer-like scaffolds have been developed with tissue regeneration in mind and therefore designed to mimic the regenerative environment of otherwise healthy tissues. However, the tumoral niche exhibits specific biophysical cues far from being 'cell friendly', suggesting that a different approach should be taken to design these artificial neoplastic niches. From bare 2D surfaces to 3D and 1D microstructured platforms, this opinion article focuses on evolving approaches used to mimic in vitro the neoplastic niche, discussing why this pathology cannot be assessed with tissue engineering (TE) approaches (i.e., using scaffolds facilitating cell growth, migration, and matrix degradation in the absence of diffusional restrictions, among others), and suggests how to improve them with recent lessons learned from mechanobiology and topobiology.

Novel approach for the assessment of ovarian follicles infiltration in polymeric electrospun patterned scaffolds.

Reproductive tissue engineering (REPROTEN) has been recently defined as the application of the tissue engineering approach targeting reproductive organs and several research works are focusing on this novel strategy. Being still an innovative field, most of the scaffold characterization techniques suitable for other tissue targets give inappropriate results, and there is the need to evaluate and investigate novel approaches. In particular the focus of this paper is the evaluation of the infiltration of ovarian follicles inside patterned electrospun scaffolds. Beyond the standard techniques, for the first time the use of magnetic resonance imaging (MRI) for this purpose is proposed and specific protocols for scaffold preparation are reported. Positive results in terms of evaluation of scaffolds incorporating follicles confirm this technique as highly effective for further applications in this field.

Facile Photochemical Modification of Silk Protein-Based Biomaterials.

Silk protein-based materials show promise for application as biomaterials for tissue engineering. The simple and rapid photochemical modification of silk protein-based materials composed of either Bombyx mori silkworm silk or engineered spider silk proteins (eADF4(C16)) is reported. Radicals formed on the silk-based materials initiate the polymerization of monomers (acrylic acid, methacrylic acid, or allylamine) which functionalize the surface of the silk materials with poly(acrylic acid) (PAA), poly(methacrylic acid) (PMAA), or poly(allylamine) (PAAm). To demonstrate potential applications of this type of modification, the polymer-modified silks are mineralized. The PAA- and PMAA-functionalized silks are mineralized with calcium carbonate, whereas the PAAm-functionalized silks are mineralized with silica, both of which provide a coating on the materials that may be useful for bone tissue engineering, which will be the subject of future investigations.

Engineered Coatings for Titanium Implants To Present Ultralow Doses of BMP-7.

The ongoing research to improve the clinical outcome of titanium implants has resulted in the implemetation of multiple approches to deliver osteogenic growth factors accelerating and sustaining osseointegration. Here we show the presentation of human bone morphogenetic protein 7 (BMP-7) adsorbed to titanium discs coated with poly(ethyl acrylate) (PEA). We have previously shown that PEA promotes fibronectin organization into nanonetworks exposing integrin- and growth-factor-binding domains, allowing a synergistic interaction at the integrin/growth factor receptor level. Here, titanium discs were coated with PEA and fibronectin and then decorated with ng/mL doses of BMP-7. Human mesenchymal stem cells were used to investigate cellular responses on these functionalized microenvironments. Cell adhesion, proliferation, and mineralization, as well as osteogenic markers expression (osteopontin and osteocalcin) revealed the ability of the system to be more potent in osteodifferentiation of the mesenchymal cells than combinations of titanium and BMP-7 in absence of PEA coatings. This work represents a novel strategy to improve the biological activity of titanium implants with BMP-7.

The size-speed-force relationship governs migratory cell response to tumorigenic factors.

Tumor development progresses through a complex path of biomechanical changes leading first to cell growth and contraction and then cell deadhesion, scattering, and invasion. Tumorigenic factors may act specifically on one of these steps or have a wider spectrum of actions, leading to a variety of effects and thus sometimes to apparent contradictory outcomes. Here we used micropatterned lines of collagen type I/fibronectin on deformable surfaces to standardize cell behavior and measure simultaneously cell size, speed of motion and magnitude of the associated traction forces at the level of a single cell. We analyzed and compared the normal human breast cell line MCF10A in control conditions and in response to various tumorigenic factors. In all conditions, a wide range of biomechanical properties was identified. Despite this heterogeneity, normal and transformed motile cells followed a common trend whereby size and contractile forces were negatively correlated with cell speed. Some tumorigenic factors, such as activation of ErbB2 or loss of the ßsubunit of casein kinase 2, shifted the whole population toward a faster speed and lower contractility state. Treatment with transforming growth factor ß induced some cells to adopt opposing behaviors such as extremely high versus extremely low contractility. Thus tumor transformation amplified preexisting population heterogeneity and led some cells to exhibit biomechanical properties that were more extreme than those observed with normal cells.

Biomineralization of Engineered Spider Silk Protein-Based Composite Materials for Bone Tissue Engineering.

Materials based on biodegradable polyesters, such as poly(butylene terephthalate) (PBT) or poly(butylene terephthalate-co-poly(alkylene glycol) terephthalate) (PBTAT), have potential application as pro-regenerative scaffolds for bone tissue engineering. Herein, the preparation of films composed of PBT or PBTAT and an engineered spider silk protein, (eADF4(C16)), that displays multiple carboxylic acid moieties capable of binding calcium ions and facilitating their biomineralization with calcium carbonate or calcium phosphate is reported. Human mesenchymal stem cells cultured on films mineralized with calcium phosphate show enhanced levels of alkaline phosphatase activity suggesting that such composites have potential use for bone tissue engineering.

Glycopolymer functionalization of engineered spider silk protein-based materials for improved cell adhesion.

Silk protein-based materials are promising biomaterials for application as tissue scaffolds, due to their processability, biocompatibility, and biodegradability. The preparation of films composed of an engineered spider silk protein (eADF4(C16)) and their functionalization with glycopolymers are described. The glycopolymers bind proteins found in the extracellular matrix, providing a biomimetic coating on the films that improves cell adhesion to the surfaces of engineered spider silk films. Such silk-based materials have potential as coatings for degradable implantable devices.

Tuning of cell-biomaterial anchorage for tissue regeneration.

Which mechanisms mediate cell attachment to biomaterials? What role does the surface charge or wettability play on cell-material anchorage? What are the currently investigated strategies to modify cell-matrix adherence spatiotemporally? Considering the development of scaffolds made of biocompatible materials to temporarily replace the structure and/or function of the extracellular matrix, focus is given to the analysis of the specific (i.e., cell adhesive peptide sequences) and unspecific (i.e., surface charge, wettability) mechanisms mediating cell-matrix interactions. Furthermore, because natural tissue regeneration is characterized by the dynamic attachment/detachment of different cell populations, the design of advanced scaffolds for tissue engineering, based in the spatiotemporal tuning of cell-matrix anchorage is discussed.

Engineered spider silk protein-based composites for drug delivery.

Silk protein-based materials are promising materials for the delivery of drugs and other active ingredients, due to their processability, biocompatibility, and biodegradability. The preparation of films composed of an engineered spider silk protein (eADF4(C16)) in combination with either a polyester (polycaprolactone) or a polyurethane (pellethane), and their physical properties are described. The release profiles are affected by both the film composition and the presence of enzymes, and release can be observed over a period of several weeks. Such silk-based composites have potential as drug eluting biocompatible coatings or implantable devices.

Cell adhesion and proliferation on RGD-modified recombinant spider silk proteins.

Due to the biocompatibility and biodegradability as well as the mechanical properties of the fibers, spider silk has become an attractive material for researchers regarding biomedical applications. In this study, the engineered recombinant spider silk protein eADF4(C16) was modified with the integrin recognition sequence RGD by a genetic (fusing the amino acid sequence GRGDSPG) as well as a chemical approach (using the cyclic peptide c(RGDfK)). Both modified silk proteins were processed into films, and thereafter characterized concerning secondary structure, water contact angle and surface roughness. No influence of the RGD-modifications on any of these film properties could be detected. However, attachment and proliferation of BALB/3T3 mouse fibroblasts were significantly improved on films made of the RGD-modified silk proteins. Interestingly, the genetically created hybrid protein (with a linear RGD sequence) showed similar or slightly better cell adhesion properties as the silk protein chemically modified with the cyclic RGD peptide.

Tuning liver stiffness against tumours: an in vitro study using entrapped cells in tumour-like microcapsules.

Liver fibrosis is a reversible pathology characterized by the up-regulated secretion and deposition of ECM proteins and inhibitors of metalloproteinases, which increase the stiffness and viscosity of this organ. Since recent studies have shown that fibrosis preceded the generation of hepatocellular carcinomas, we hypothesize that liver fibrosis could play a role as a mechanism for restricting uncontrolled cell proliferation, inducing the mortality of cancer cells and subsequent development of primary tumours. With this purpose, in this work we analysed in vitro how the modulation of stiffness can influence proliferation, viability and aggregation of hepatocarcinoma cells (HepG(2)) embedded in 3D micromilieus mimicking values of elasticity of fibrotic liver tissues. Experiments were performed by immobilizing up to 10 HepG(2) cells within microcapsules made of 0.8%, 1.0% and 1.4% w/v alginate which, besides having values of elasticity from the lower-healthy to the upper-fibrotic range liver tissues, lacked domains for proteases, mimicking the micromilieu existing in hepatic primary tumours. Our results show that entrapped cells exhibited a short duplication phase followed by an irreversible decay stage, in which cell mortality could be mediated by two mechanisms: mechanical stress, in the case of cells entrapped in a stiffer micromilieu; and mass transfer limitations produced by pore coarsening at the interface cell-matrix, in softer micromilieus. According to the authors' knowledge, this work represents the first attempt to elucidate the role of liver fibrosis during Hepatocarcinoma pathologies, suggesting that the generation of a non-biodegradable and mechanically unfavourable environment surrounding cancer cells could control the proliferation, migration of metastatic cells and the subsequent development of primary tumours.

Determination of pore size distribution at the cell-hydrogel interface.

BACKGROUND: Analyses of the pore size distribution in 3D matrices such as the cell-hydrogel interface are very useful when studying changes and modifications produced as a result of cellular growth and proliferation within the matrix, as pore size distribution plays an important role in the signaling and microenvironment stimuli imparted to the cells. However, the majority of the methods for the assessment of the porosity in biomaterials are not suitable to give quantitative information about the textural properties of these nano-interfaces. FINDINGS: Here, we report a methodology for determining pore size distribution at the cell-hydrogel interface, and the depth of the matrix modified by cell growth by entrapped HepG(2) cells in microcapsules made of 0.8% and 1.4% w/v alginate. The method is based on the estimation of the shortest distance between two points of the fibril-like network hydrogel structures using image analysis of TEM pictures. Values of pore size distribution determined using the presented method and those obtained by nitrogen physisorption measurements were compared, showing good agreement. A combination of these methodologies and a study of the cell-hydrogel interface at various cell culture times showed that after three days of culture, HepG(2) cells growing in hydrogels composed of 0.8% w/v alginate had more coarse of pores at depths up to 40 nm inwards (a phenomenon most notable in the first 20 nm from the interface). This coarsening phenomenon was weakly observed in the case of cells cultured in hydrogels composed of 1.4% w/v alginate. CONCLUSIONS: The method purposed in this paper allows us to obtain information about the radial deformation of the hydrogel matrix due to cell growth, and the consequent modification of the pore size distribution pattern surrounding the cells, which are extremely important for a wide spectrum of biotechnological, pharmaceutical and biomedical applications.

Silk-based materials for biomedical applications.

Since the beginning of civilization, humans have exploited nature as an extraordinary source of materials for medical applications. Most natural materials comprise biopolymers such as nucleic acids and protein-polysaccharides. For biomedical applications, proteins such as collagens have been traditionally employed. Other proteins are silk fibres produced by arthropods (e.g. silkworms and spiders), which provide interesting mechanical properties and the absence of toxicity. Silks present almost all characteristics desirable for biomedical applications, but the research on the underlying proteins has only recently commenced. In the present review, we summarize the current research related to silk being used as a material for cell culture and tissue engineering, particularly focusing on cell-surface adherence, mechanical and textural properties, toxicity, immunogenicity and biodegradability.

Determination of the decay rate constant for hepatocytes immobilized in alginate microcapsules.

Primary mouse hepatocytes (between 10-250 cells per capsule) were immobilized within 1.0% w/v alginate microbeads. The textural properties of the alginate matrix were characterized and a full protocol based upon the measurement of the initial rate of Resazurin reduction was studied and standardized. Using this method, the decay rate constant (K(d) = 0.45 +/- 0.01 days(-1)) and the time in which the cell viability decreases in half (VI(50) = 37 +/- 0.7 h) have been measured. The method was compared with the analysis of cell vitality using Calcein A/M and Ethidium Homodimer I. Differences between the two methods were found in the viability profile due to the significant presence of double stained cells along the culture time. According to the author's knowledge, this is the first report of a systematic study and determination of the K(d) value for immobilized hepatocytes, incorporating a wide range of cell concentrations within the alginate matrix.