Co-culture of human induced pluripotent stem cell-derived retinal pigment epithelial cells and endothelial cells on double collagen-coated honeycomb films.

In vitro cell culture models representing the physiological and pathological features of the outer retina are urgently needed. Artificial tissue replacements for patients suffering from degenerative retinal diseases are similarly in great demand. Here, we developed a co-culture system based solely on the use of human induced pluripotent stem cell (hiPSC)-derived cells. For the first time, hiPSC-derived retinal pigment epithelium (RPE) and endothelial cells (EC) were cultured on opposite sides of porous polylactide substrates prepared by breath figures (BF), where both surfaces had been collagen-coated by Langmuir-Schaefer (LS) technology. Small modifications of casting conditions during material preparation allowed the production of free-standing materials with distinct porosity, wettability and ion diffusion capacity. Complete pore coverage was achieved by the collagen coating procedure, resulting in a detectable nanoscale topography. Primary retinal endothelial cells (ACBRI181) and umbilical cord vein endothelial cells (hUVEC) were utilised as EC references. Mono-cultures of all ECs were prepared for comparison. All tested materials supported cell attachment and growth. In mono-culture, properties of the materials had a major effect on the growth of all ECs. In co-culture, the presence of hiPSC-RPE affected the primary ECs more significantly than hiPSC-EC. In consistency, hiPSC-RPE were also less affected by hiPSC-EC than by the primary ECs. Finally, our results show that the modulation of the porosity of the materials can promote or prevent EC migration. In short, we showed that the behaviour of the cells is highly dependent on the three main variables of the study: the presence of a second cell type in co-culture, the source of endothelial cells and the biomaterial properties. The combination of BF and LS methodologies is a powerful strategy to develop thin but stable materials enabling cell growth and modulation of cell-cell contact. STATEMENT OF SIGNIFICANCE: Artificial blood-retinal barriers (BRB), mimicking the interface at the back of the eye, are urgently needed as physiological and disease models, and for tissue transplantation targeting patients suffering from degenerative retinal diseases. Here, we developed a new co-culture model based on thin, biodegradable porous films, coated on both sides with collagen, one of the main components of the natural BRB, and cultivated endothelial and retinal pigment epithelial cells on opposite sides of the films, forming a three-layer structure. Importantly, our hiPSC-EC and hiPSC-RPE co-culture model is the first to exclusively use human induced pluripotent stem cells as cell source, which have been widely regarded as an practical candidate for therapeutic applications in regenerative medicine.

Effect of Melt-Derived Bioactive Glass Particles on the Properties of Chitosan Scaffolds.

This study reports on the processing of three-dimensional (3D) chitosan/bioactive glass composite scaffolds. On the one hand, chitosan, as a natural polymer, has suitable properties for tissue engineering applications but lacks bioactivity. On the other hand, bioactive glasses are known to be bioactive and to promote a higher level of bone formation than any other biomaterial type. However, bioactive glasses are hard, brittle, and cannot be shaped easily. Therefore, in the past years, researchers have focused on the processing of new composites. Difficulties in reaching composite materials made of polymer (synthetic or natural) and bioactive glass include: (i) The high glass density, often resulting in glass segregation, and (ii) the fast bioactive glass reaction when exposed to moisture, leading to changes in the glass reactivity and/or change in the polymeric matrix. Samples were prepared with 5, 15, and 30 wt% of bioactive glass S53P4 (BonAlive ®), as confirmed using thermogravimetric analysis. MicrO-Computed tomography and optical microscopy revealed a flaky structure with porosity over 80%. The pore size decreased when increasing the glass content up to 15 wt%, but increased back when the glass content was 30 wt%. Similarly, the mechanical properties (in compression) of the scaffolds increased for glass content up to 15%, but decreased at higher loading. Ions released from the scaffolds were found to lead to precipitation of a calcium phosphate reactive layer at the scaffold surface. This is a first indication of the potential bioactivity of these materials. Overall, chitosan/bioactive glass composite scaffolds were successfully produced with pore size, machinability, and ability to promote a calcium phosphate layer, showing promise for bone tissue engineering and the mechanical properties can justify their use in non-load bearing applications.

Characterization of the microstructure of hydrazone crosslinked polysaccharide-based hydrogels through rheological and diffusion studies.

Microstructure plays an essential role in the control of hydrogel properties. It is also an important factor when cells or drugs are encapsulated inside the hydrogel. In this work, the microstructures of hydrazone crosslinked hyaluronan-, alginate- and gellan gum-based hydrogels were evaluated thoroughly for the first time by using rheology- and diffusion (fluorescence recovery after photobleaching, FRAP)-based methods. The effect of gel parameters on the viscoelastic and diffusion properties of hydrogels, and further on their structural parameters (mesh size, average molecular weight of the polymer chain between neighboring crosslinks, crosslinking density) are shown. Results further show that diffusivity decreased when larger dextran sizes were used, which were equivalent to the mesh sizes of hydrogels (15 nm to 47 nm) evaluated by the rheological method. This mesh size range allows the transportation of smaller molecules, but also peptides and most of the proteins. A correlation between the storage modulus and the structural parameters was also shown. Overall, hydrazone crosslinking offers an easy way to produce polysaccharide-based hydrogels with variable microstructures by altering the gel parameters.

Breath figures in tissue engineering and drug delivery: State-of-the-art and future perspectives.

The breath figure (BF) method is an easy, low-cost method to prepare films with a highly organized honeycomb-like porous surface. The particular surface topography and porous nature of these materials makes them valuable substrates for studying the complex effects of topography on cell fate, and to produce biomimetic materials with high performance in tissue engineering. Numerous researchers over the last two decades have studied the effects of the honeycomb topography on a variety of primary and immortalized cell lines, and drew important conclusions that can be translated to the construction of optimal biomaterials for cell culture. The literature also encouragingly shows the potential of honeycomb films to induce differentiation of stem cells down a specific lineage without the need for biochemical stimuli. Here, we review the main studies where BF honeycomb films are used as substrates for tissue engineering applications. Furthermore, we highlight the numerous advantages of the porous nature of the films, such as the enhanced, spatially controlled adsorption of proteins, the topographical cues influencing cellular behavior, and the enhanced permeability which is essential both in vitro and in vivo. Finally, this review highlights the elegant use of honeycomb films as drug-eluting biomaterials or as reservoirs for distinct drug delivery systems. STATEMENT OF SIGNIFICANCE: Combining biocompatible surfaces and 3D nano/microscale topographies, such as pores or grooves, is an effective strategy for manufacturing tissue engineering scaffolds. The breath figure (BF) method is an easy technique to prepare cell culture substrates with an organized, honeycomb-like porous surface. These surface features make these scaffolds valuable for studying how the cells interact with the biomaterials. Their unique surface topography can also resemble the natural environment of the tissues in the human body. For that reason, numerous studies, using different cell types, have shown that honeycomb films can constitute high performance substrates for cell culture. Here, we review those studies, we highlight the advantages of honeycomb films in tissue engineering and we discuss their potential as unique drug-eluting systems.

Langmuir-Schaefer film deposition onto honeycomb porous films for retinal tissue engineering.

Age-related macular degeneration (AMD) is the leading cause of vision loss in senior citizens in the developed world. The disease is characterised by the degeneration of a specific cell layer at the back of the eye - the retinal pigment epithelium (RPE), which is essential in retinal function. The most promising therapeutic option to restore the lost vision is considered to be RPE cell transplantation. This work focuses on the development of biodegradable biomaterials with similar properties to the native Bruch's membrane as carriers for RPE cells. In particular, the breath figure (BF) method was used to create semi-permeable microporous films, which were thereafter used as the substrate for the consecutive Langmuir-Schaefer (LS) deposition of highly organised layers of collagen type I and collagen type IV. The newly developed biomaterials were further characterised in terms of surface porosity, roughness, hydrophilicity, collagen distribution, diffusion properties and hydrolytic stability. Human embryonic stem cell-derived RPE cells (hESC-RPE) cultured on the biomaterials showed good adhesion, spreading and morphology, as well as the expression of specific protein markers. Cell function was additionally confirmed by the assessment of the phagocytic capacity of hESC-RPE. Throughout the study, microporous films consistently showed better results as cell culture materials for hESC-RPE than dip-coated controls. This work demonstrates the potential of the BF-LS combined technologies to create biomimetic prosthetic Bruch's membranes for hESC-RPE transplantation. STATEMENT OF SIGNIFICANCE: Age-related macular degeneration (AMD) is a leading cause of central blindness in developed countries, associated with the degeneration of the retinal pigment epithelium (RPE), a specific cell layer at the back of the eye. Transplantation of RPE cells derived from stem cells is considered the best option to treat these patients. In this work, we developed a cell carrier for human embryonic stem cell-derived RPE that resembled the upper layers of the membrane that naturally supports the RPE cells in the retina. The new combination of technologies employed in this study resulted in very promising materials as confirmed by our studies on cell proliferation, morphology and function.

Honeycomb porous films as permeable scaffold materials for human embryonic stem cell-derived retinal pigment epithelium.

Age-related macular degeneration (AMD) is a leading cause of blindness in developed countries, characterised by the degeneration of the retinal pigment epithelium (RPE), a pigmented cell monolayer that closely interacts with the photoreceptors. RPE transplantation is thus considered a very promising therapeutic option to treat this disease. In this work, porous honeycomb-like films are for the first time investigated as scaffold materials for human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE). By changing the conditions during film preparation, it was possible to produce films with homogeneous pore distribution and adequate pore size (∼3-5 µm), that is large enough to ensure high permeability but small enough to enable cell adherence and spreading. A brief dip-coating procedure with collagen type IV enabled the homogeneous adsorption of the protein to the walls and bottom of pores, increasing the hydrophilicity of the surface. hESC-RPE adhered and proliferated on all the collagen-coated materials, regardless of small differences in pore size. The differentiation of hESC-RPE was confirmed by the detection of specific RPE protein markers. These results suggest that the porous honeycomb films can be promising candidates for hESC-RPE tissue engineering, importantly enabling the free flow of ions and molecules across the material. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1646-1656, 2016.

Sustained release of naltrexone from poly(n-isopropylacrylamide) microgels.

The release of the opioid antagonist naltrexone from neutral poly(N-isopropylacrylamide) (PNIPAAM) microgels and negatively charged PNIPAAM microgels containing acrylic acid groups (PNIPAAM-co-PAA) has been studied at various microgel and drug concentrations. The release curves were found to be well represented by the Weibull equation. The release rates were observed to be dependent on the microgel concentration. At most conditions, the release from the charged microgels was slower than for the neutral microgels. In addition, the charged microgels exhibited a release lag time, which was dependent on the microgel concentration. No significant lag time could be observed for the neutral microgels. Increasing the naltrexone concentration did not significantly affect the release rates from the neutral microgels, but the release from the charged microgels became faster. The microgels did not exhibit any significant cytotoxic effect on HeLa cells at the tested concentrations.

Thermoresponsive polymers as gene and drug delivery vectors: architecture and mechanism of action.

INTRODUCTION: This topic is important as it allows for improved specificity in drug delivery, providing possibilities for reduced side effects, and thereby improved pharmacotherapy. As a wealth of different polymers and mechanisms of action has been suggested, a systematic overview of the field is of current importance. AREAS COVERED: This article presents an overview over a selection of thermoresponsive polymers suitable as excipients in systems for gene and drug delivery with particular emphasis on the influence of polymer structure, composition, molecular weight (MW) and architecture on the responsive mechanisms. Due to the immense number of reports on these increasingly popular materials, focus has been restricted to the use of micelle-forming polymers with a lower critical solution temperature, temperature-responsive hydrogels for drug delivery applications and temperature-sensitive polymers as non-viral vectors for polynucleotide delivery. Specific examples covered are poly-(N-isopropylacrylamide) (PNIPAAM), Pluronics and their derivatives. It is concluded that the studies constitute an excellent platform for development of thermoresponsive systems with improved gene and drug delivery properties. EXPERT OPINION: A thorough knowledge of factors important for loading efficiency and drug release is necessary to be able to develop optimal nano-carriers for the future. Other issues that are not fully understood is how small the carriers need to be, and which manufacturing procedures should be used.

Temperature-responsive cationic block copolymers as nanocarriers for gene delivery.

Cationic block copolymers have been regarded as promising alternatives to the use of viral vectors for gene delivery. In this work, poly(N-isopropylacrylamide)n-block-poly((3-acrylamidopropyl)trimethylammonium chloride)m (PNIPAAMn-b-PAMPTMA(+)m) block copolymers with n=48 or 65 and m=6, 10 or 20 were synthesized and evaluated in terms of their potential for in vitro transfection of HeLa cells. These block copolymers collapse above a phase transition temperature, allowing the entrapment of the DNA molecules they are adsorbed to. The transfection efficiency increased with polymer concentration and was higher in the presence of a long PNIPAAM block and for a short charged block. In general, increasing the length of the charged block decreased the transfection efficiency. Additionally, polymer-DNA complexes (polyplexes) formed at lower polymer/DNA charge ratios caused lower cell toxicity levels. All polymers were shown to efficiently protect the DNA, even when they were present at low concentrations. At 37°C, the polyplexes mostly formed structures with size ranging from 100 to 500nm. The results also showed that the thermoresponsive contraction of PNIPAAM causes the charged block segments to be pressed out to the surface. The formation of compact structures seems to be a key factor in achieving high transfection efficiency.

In vitro cytotoxicity of a thermoresponsive gel system combining ethyl(hydroxyethyl) cellulose and lysine-based surfactants.

The cytotoxicity of three lysine-derived surfactants with a gemini-like structure was evaluated on HeLa cells. The half maximal effective concentration (EC(50)) was estimated from the dose-response curves and the values indicated an increase in toxicity with the increase in alkyl chain length. The shorter chain length surfactant (C(6)) was shown to be less cytotoxic than sodium dodecyl sulfate (SDS), and all the lysine-derived surfactants were less toxic than the cationic cetyl trimethylammonium bromide (CTAB). The presence of ethyl (hydroxyethyl) cellulose (EHEC), shown previously to form thermoresponsive gels in combination with these surfactants, was found to contribute to a lower toxicity on HeLa cells. The conjecture is that the polymer-surfactant interactions in forming mixed micelles are the key contributors to the enhanced biocompatibility of the hydrogels. The most promising results were obtained in the presence of either the most hydrophilic surfactant or in the presence of the longest chain-length surfactant. For the latter, very low concentrations are needed to induce a sol-gel transition of EHEC semi-dilute solutions.

Thermoresponsive hydrogels with low toxicity from mixtures of ethyl(hydroxyethyl) cellulose and arginine-based surfactants.

Ethyl(hydroxyethyl) cellulose (EHEC) is known to form hydrogels in water at elevated temperatures in the presence of an ionic surfactant. In this paper, the potential use of arginine-based surfactants is explored considering the production of a low toxicity thermoresponsive hydrogel for pharmaceutical and biomedical applications. The interactions between EHEC and the monomeric surfactant N(α)-lauroyl-L-arginine methyl ester (LAM) and two gemini surfactants N(α),N(ω)-bis(N(α)-acylarginine) α,ω-dialkyl amides were evaluated by Rheo-Small Angle Light Scattering measurements. The complex viscosity of the systems was dependent on surfactant concentration and temperature. Under specific conditions, soft gels of homogeneous structure were produced. The cloud point (CP) of the EHEC-LAM system varied significantly with surfactant concentration, while only moderate CP changes were found in the presence of the gemini surfactants. Finally, the effect of the surfactants on the viability of a human cell line was evaluated. Despite the lower toxicity of LAM, the superior gel forming efficiency of the gemini surfactants at lower concentrations revealed their advantageous suitability as components of a biocompatible thermoresponsive gel system.