Transfer of fibroblast sheets cultured on thermoresponsive dishes with membranes.

In cell or tissue engineering, it is essential to develop a support for cell-to-cell adhesion, which leads to the generation of cell sheets connected by extracellular matrix. Such supports must be hydrophobic and should result in a detachable cell sheet. A thermoresponsive support that enables the cultured cell sheet to detach using only a change in temperature could be an interesting alternative in regenerative medicine. The aim of this study was to evaluate plates covered with thermoresponsive polymers as supports for the formation of fibroblast sheets and to develop a damage-free procedure for cell sheet transfer with the use of membranes as transfer tools. Human skin fibroblasts were seeded on supports coated with a thermoresponsive polymer: commercial UpCell™ dishes (NUNC™) coated with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and dishes coated with thermoresponsive poly(tri(ethylene glycol) monoethyl ether methacrylate) (P(TEGMA-EE)). Confluent fibroblast sheets were effectively cultured and harvested from both commercial PNIPAM-coated dishes and laboratory P(TEGMA-EE)-coated dishes. To transfer a detached cell sheet, two membranes, Immobilon-P(®) and SUPRATHEL(®), were examined. The use of SUPRATHEL for relocating the cell sheets opens a new possibility for the clinical treatment of wounds. This study established the background for implementing thermoresponsive supports for transplanting in vitro cultured fibroblasts.

Controlling the Crystallinity of Thermoresponsive Poly(2-oxazoline)-Based Nanolayers to Cell Adhesion and Detachment.

Semicrystalline, thermoresponsive poly(2-isopropyl-2-oxazoline) (PIPOx) layers covalently bonded to glass or silica wafers were obtained via the surface-termination of the living polymer chains. Polymer solutions in acetonitrile were exposed to 50 °C for various time periods and were poured onto the functionalized solid wafers. Fibrillar crystallites formed in polymerization solutions settled down onto the wafers next to the amorphous polymer. The amount of crystallites adsorbed on thermoresponsive polymer layers depended on the annealing time of the PIPOx solution. The wettability of PIPOx layers decreased with the increasing amount of crystallites. The higher content of crystallites weakened the temperature response of the layer, as evidenced by the philicity and thickness measurements. Semicrystalline thermoresponsive PIPOx layers were used as biomaterials for human dermal fibroblasts (HDFs) culture and detachment. The presence of crystallites on the PIPOx layers promoted the proliferation of HDFs. Changes in the physicochemical properties of the layer, caused by the temperature response of the polymer, led to the change in the cells shape from a spindle-like to an ellipsoidal shape, which resulted in their detachment. A supporting membrane was used to assist the detachment of the cells from PIPOx biosurfaces and to prevent the rolling of the sheet.

Poly(2-substituted-2-oxazoline) surfaces for dermal fibroblasts adhesion and detachment.

The thermoresponsive surfaces of brush structure (linear polymer chains tethered on the surface) based on poly(2-isopropyl-2-oxazoline)s and copolymers of 2-ethyl-2-oxazoline and 2-nonyl-2-oxazoline were obtained using the grafting-to method. The living oxazoline (co)polymers have been synthesized by cationic ring-opening polymerization and subsequently terminated by the reactive amine groups present on the surface. The changes in the surface morphology, philicity and thickness occurring during surface modification were monitored via atomic force microscopy, contact angle and ellipsometry. The thickness of the (co)poly(2-substituted-2-oxazoline) layers ranged from 4 to 11 nm depending on the molar mass of immobilized polymer and reversibly varied with the temperature changes. This confirmed thermoresponsive properties of obtained surfaces. The obtained polymer surfaces were used as a support for dermal fibroblast culture and detachment. The fibroblasts' adhesion and proliferation on the polymer surfaces were observed when the culture temperature was above the cloud point temperature of the immobilized polymer. Lowering the temperature resulted in the detachment of the dermal fibroblast sheets from the polymer layers, which makes these surfaces suitable for the treatment of wounds and in skin tissue engineering.

Degradable Nanogels Based on Poly[Oligo(Ethylene Glycol) Methacrylate] (POEGMA) Derivatives through Thermo-Induced Aggregation of Polymer Chain and Subsequent Chemical Crosslinking.

Polymer nanogels-considered as nanoscale hydrogel particles-are attractive for biological and biomedical applications due to their unique physicochemical flexibility. However, the aggregation or accumulation of nanoparticles in the body or the occurrence of the body's defense reactions still pose a research challenge. Here, we demonstrate the fabrication of degradable nanogels using thermoresponsive, cytocompatible poly[oligo(ethylene glycol) methacrylate]s-based copolymers (POEGMA). The combination of POEGMA's beneficial properties (switchable affinity to water, nontoxicity, non-immunogenicity) along with the possibility of nanogel degradation constitute an important approach from a biological point of view. The copolymers of oligo(ethylene glycol) methacrylates were partially modified with short segments of degradable oligo(lactic acid) (OLA) terminated with the acrylate group. Under the influence of temperature, copolymers formed self-assembled nanoparticles, so-called mesoglobules, with sizes of 140-1000 nm. The thermoresponsive behavior of the obtained copolymers and the nanostructure sizes depended on the heating rate and the presence of salts in the aqueous media. The obtained mesoglobules were stabilized by chemical crosslinking via thiol-acrylate Michael addition, leading to nanogels that degraded over time in water, as indicated by the DLS, cryo-TEM, and AFM measurements. Combining these findings with the lack of toxicity of the obtained systems towards human fibroblasts indicates their application potential.

Polymeric Carriers for Delivery Systems in Biomedical Applications-In Memory of Professor Andrzej Dworak.

Since the appearance of the first civilizations, various substances have been used to improve human health [...].

Laminar Biomaterial Composite of PVA Cryogel with Amnion as Potential Wound Dressing.

Gel dressings, composed of polymers both natural and synthetic, are successfully used in the treatment of burn wounds. They protect the burn wound site against adverse external factors, ensure an adequate level of tissue hydration, have soothing and pain-relieving properties, and also support the healing process and reduce the risk of pathological scars. Another promising material that can be used in the wound-healing process is an amnion membrane. Due to its valuable properties such as protecting the body against bacterial infections and permeability to nutrition, it has found usage in different brands of medicine. In this work, we have combined the beneficial properties of hydrogels and amnion in order to make the laminar dressing that may serve for wound healing. For that purpose, the physically crosslinked cryogel of poly(vinyl alcohol) (PVA) was covered with an amnion membrane. Subsequently, gamma irradiation was performed, leading to the simultaneous internal crosslinking of the hydrogel, its permanent bonding with the amnion, and dressing sterilization. The physicochemical properties of the dressing including gel fraction, swelling, and hardness were studied. Biological tests such as the MTT assay, antimicrobial activity, and histopathological examination confirmed that the obtained material constituted a promising candidate for further, more in-depth studies aiming at wound dressing application.

Influence of electron beam irradiation on extracellular matrix of the human allogeneic skin grafts.

The nonviable allogeneic human skin grafts might be considered as the most suitable skin substitutes in the treatment of extensive and deep burns. However, in accordance to biological security such grafts require the final sterilization prior to clinical application. The aim of the study was to verify the influence of electron beam irradiation of three selected doses: 18, 25, and 35 kGy on the extracellular matrix of human skin. Prior to sterilization, the microbiological tests were conducted and revealed contamination in all examined cases. Individual groups were subjected to single electron beam radiation sterilization at proposed doses and then subjected to microbiological tests again. The results of microbiological testing performed for all irradiation doses used were negative. Only in the control group was a growth of microorganisms observed. The FTIR spectrometry tests were conducted followed by the histological evaluation and mechanical tests. In addition, cost analysis of radiation sterilization of individual doses was performed. The results of spectroscopic analysis, mechanical tests, and histological staining showed no significant changes in composition and characteristics of tested tissues after their irradiation, in comparison to control samples. The cost analysis has shown that irradiation with 18 kGy is the most cost-effective and 35 kGy is the least favorable. However, according to biological risk reduction, the recommended sterilization dose is 35 kGy, despite the higher price compared to the other doses tested.

The Role of Polymer Structure in Formation of Various Nano- and Microstructural Materials: 30 Years of Research in the Laboratory of Nano- and Microstructural Materials at the Centre of Polymer and Carbon Materials PAS.

The review summarizes the research carried out in the Laboratory of Nano- and Microstructural Materials at the Centre of Polymer and Carbon Materials, Polish Academy of Sciences (CMPW PAS). Studies carried out for many years under the guidance of Professor Andrzej Dworak led to the development and exploration of the mechanisms of oxirane and cyclic imine polymerization and controlled radical polymerization of methacrylate monomers. Based on that knowledge, within the last three decades, macromolecules with the desired composition, molar mass and topology were obtained and investigated. The ability to control the structure of the synthesized polymers turned out to be important, as it provided a way to tailor the physiochemical properties of the materials to their specific uses. Many linear polymers and copolymers as well as macromolecules with branched, star, dendritic and hyperbranched architectures were synthesized. Thanks to the applied controlled polymerization techniques, it was possible to obtain hydrophilic, hydrophobic, amphiphilic and stimulus-sensitive polymers. These tailor-made polymers with controlled properties were used for the construction of various types of materials, primarily on the micro- and nanoscales, with a wide range of possible applications, mainly in biomedicine. The diverse topology of polymers, and thus their properties, made it possible to obtain various types of polymeric nanostructures and use them as nanocarriers by encapsulation of biologically active substances. Additionally, polymer layers were obtained with features useful in medicine, particularly regenerative medicine and tissue engineering.

Selective Partial Hydrolysis of 2-isopropyl-2-oxazoline Copolymers towards Decreasing the Ability to Crystallize.

Poly(2-isopropyl-2-oxazoline) (PiPrOx) is readily prone to crystallization both in solid and from solutions. This feature is detrimental for certain applications. Here, we examine whether the presence of unsubstituted ethyleneimine (EI) units, a gradient distributed within a polymer chain composed of 2-isopropyl-2-oxazoline (iPrOx) and 2-methyl-2-oxazoline (MOx) units, decreases the ability to crystallize the copolymer and affects thermal properties compared to the homopolymer of iPrOx. We assumed that the separation of stiff iPrOx units by the more flexible EI will affect the spatial arrangements of the ordered chains, slightly plasticize and, as a result, decrease their ability to crystallize. The selective hydrolysis of gradient iPrOx and 2-methyl-2-oxazoline (MOx) copolymers, carried out under mild conditions, led to iPrOx/MOx/EI copolymers. To the best of our knowledge, the selective hydrolysis of these copolymers has never been carried out before. Their thermal properties and crystallization abilities, both in a solid state and from an aqueous solution, were analyzed. Based on the analysis of polymer charge and cytotoxicity studies, the potential use of the copolymers obtained was indicated in some biological systems.

Thermoresponsive Nanogels of Modified Poly((di(ethylene glycol) methyl ether methacrylate)-co-(2-aminoethyl methacrylate))s.

A series of copolymers of di(ethylene glycol) methyl ether methacrylate (D) and 2-aminoethyl methacrylate (A) (P(D-co-A)) with variable ratios of comonomers were synthesized using atom transfer radical polymerization. Then, the amino groups of obtained copolymers were modified to clickable azide or prop-2-yn-1-yl carbamate groups. A thermoresponsive copolymers were obtained with the value of cloud point temperature (TCP) dependent on the type and number of functional groups in the copolymer and on the concentration of solutions. For P(D-co-A) copolymers, the TCP increased with increasing content of 2-aminoethyl methacrylate comonomer. The presence of azide and prop-2-yn-1-yl carbamate groups caused the changes of TCP of modified copolymers. All studied copolymers in dilute aqueous solutions aggregated above TCP to nanoparticles with sizes dependent on the solution concentration, heating procedures, and types and numbers of functional groups present in a copolymer chain. The presence of hydrophilic elements in the chain and the increase in the copolymer concentration led to the enlargement of the particle sizes. Aggregates were crosslinked using click reaction between an azide and prop-2-yn-1-yl carbamate groups that led to stable thermoresponsive nanogels. A systematic study of the behavior of copolymers allowed the determination of the chains useful for possible application in drug delivery.

Antimicrobial Activity of Hybrid Nanomaterials Based on Star and Linear Polymers of N,N'-Dimethylaminoethyl Methacrylate with In Situ Produced Silver Nanoparticles.

Well-defined linear and multi-arm star polymer structures were used as the templates for in situ synthesis and stabilization of silver nanoparticles (AgNPs). This approach led to hybrid nanomaterials with high stability and antibacterial activity to both Gram-positive and Gram-negative bacterial strains. The ecologically friendly so called "green" synthesis of nanomaterials was performed through AgNPs preparation in the aqueous solutions of star and linear poly(N,N'-dimethylaminoethyl methacrylate)s (PDMAEMAs); the process was followed with time. The size, shape, and zeta potential of the obtained hybrids were determined. To our knowledge, this is the first time that the antibacterial activity of PDMAEMA hybrid nanomaterial against Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa was investigated and assessed by minimum inhibitory concentration (MIC) and minimum biocidal concentration (MBC). Completely quaternized with ethyl bromide, star and linear PDMAEMAs were used in comparative biological tests. The modification of the polymers with in situ-formed AgNPs increased the antibacterial properties against all studied strains of bacteria by several times in comparison to non-modified polymers and quaternized polymers. These results yield novel nanohybrid materials that can be useful for applications in medicine and biology.

Processing of (Co)Poly(2-oxazoline)s by Electrospinning and Extrusion from Melt and the Postprocessing Properties of the (Co)Polymers.

Poly(2-oxazoline) (POx) matrices in the form of non-woven fibrous mats and three-dimensional moulds were obtained by electrospinning and fused deposition modelling (FDM), respectively. To obtain these materials, poly(2-isopropyl-2-oxazoline) (PiPrOx) and gradient copolymers of 2-isopropyl- with 2-n-propyl-2-oxazoline (P(iPrOx-nPrOx)), with relatively low molar masses and low dispersity values, were processed. The conditions for the electrospinning of POx were optimised for both water and the organic solvent. Also, the FDM conditions for the fabrication of POx multi-layer moulds of cylindrical or cubical shape were optimised. The properties of the POx after electrospinning and extrusion from melt were determined. The molar mass of all (co)poly(2-oxazoline)s did not change after electrospinning. Also, FDM did not influence the molar masses of the (co)polymers; however, the long processing of the material caused degradation and an increase in molar mass dispersity. The thermal properties changed significantly after processing of POx what was monitored by increase in enthalpy of exo- and endothermic peaks in differential scanning calorimetry (DSC) curve. The influence of the processing conditions on the structure and properties of the final material were evaluated having in a mind their potential application as scaffolds.

Poly(2-oxazoline) Matrices with Temperature-Dependent Solubility-Interactions with Water and Use for Cell Culture.

In this work, we studied the stability of matrices with temperature-dependent solubility and their interactions with water at physiological temperature for their application in cell culture in vitro. Gradient copolymers of 2-isopropyl- with 2-n-propyl-2-oxazoline (P(iPrOx-nPrOx)) were used to prepare the matrices. The comonomer ratio during polymerization was chosen such that the cloud point temperature (TCP) of the copolymer was below 37 °C while the glass transition (Tg) was above 37 °C. The role of the support for matrices in the context of their stability in aqueous solution was examined. Therefore, matrices in the form of both self-supported bulk polymer materials (fibrillar mats and molds) and polymer films supported on the silica slides were examined. All of the matrices remained undissolved when incubated in water at a temperature above TCP. For the self-supported mats and molds, we observed the loss of shape stability, but, in the case of films supported on silica slides, only slight changes in morphology were observed. For a more in-depth investigation of the origin of the shape deformation of self-supported matrices, we analyzed the wettability, thickness, and water uptake of films on silica support because the matrices remained undeformed under these conditions. It was found that, above the TCP of P(iPrOx-nPrOx), the wettability of the films decreased, but at the same time the films absorbed water and swelled. We examined how this specific behavior of the supported films influenced the culture of fibroblasts. The temperature-dependent solubility of the matrices and the possibility of noninvasive cell separation were also examined.

Fibroblast and keratinocyte crosstalk: the effect of a poly(tri[ethylene glycol] ethyl ether methacrylate) thermoresponsive surface on short-term co-culture.

The treatment of difficult-to-treat wounds can be challenging. Although a number of approaches have been investigated, the healing process may be slow and unsatisfactory. An alternative approach is the use of a continuous sheet of skin cells applied over a wound which may improve cell implantation and patient recovery. To analyse the gene expression profile of fibroblast/keratinocyte co-culture on poly(tri[ethylene glycol] ethyl ether methacrylate) (P[TEGMA-EE]), a thermoresponsive biocompatible surface. Cultures were grown for 72 hours as a continuous layer on P(TEGMA-EE). Assays for genotoxicity, cell morphology, and fluorescence-assisted flow cytometry were performed to exclude adverse effects. A gene expression profile related to the extracellular matrix was investigated by microarray analysis. For fibroblast monocultures and fibroblast/keratinocyte co-cultures maintained for 72 hours on P(TEGMA-EE), no change in morphology or specific surface markers, or DNA damage (comet assay) was observed, relative to control surface. Moreover, no detrimental impact was ascertained based on microarray analysis. In response to lowered temperature, the detachment of a continuous cell layer sheet from the thermoresponsive surface was observed. When gene expression was compared between fibroblasts cultured alone and co-cultured with keratinocytes on P(TEGMA-EE), 10 genes were shown to be differentially expressed. Of these genes, six were significantly differentially expressed between cultures grown on P(TEGMA-EE) and human skin samples. Our results indicate that P(TEGMA-EE) is fully biocompatible and is therefore a suitable surface for successful preparation and recovery of two-layered fibroblast/keratinocyte co-culture as a continuous sheet of cells.

Photocrosslinking of Polyglycidol and Its Derivative: Route to Thermoresponsive Hydrogels.

Hydrogels of biologically well-tolerated, high-molar-mass polyglycidol (PGl) and its thermoresponsive derivative poly(glycidol-co-ethyl glycidyl carbamate) have been obtained by direct UV crosslinking in the solid state. Polymers with molar masses up to 1.45 × 106 g mol-1 were crosslinked in the presence of benzophenone or (4-benzoylbenzyl)trimethylammonium chloride as photosensitizers. The photosensitizer concentration was varied from 2 to 10 wt%. The influence of polymer composition and photosensitizer type and amount on the crosslinking efficiency, swelling and temperature behavior of the obtained hydrogels was investigated. The photocrosslinking of PGl and poly(glycidol-co-ethyl glycidyl carbamate) led to hydrogels with swelling degrees up to 1700%. The swelling degrees of the hydrogels decreased with the increase of the environmental temperature indicating the thermoresponsive nature of gels. The swelling of obtained gels can be controlled by varying the composition of the copolymer precursor and by the network density.

Poly[tri(ethylene glycol) ethyl ether methacrylate]-coated surfaces for controlled fibroblasts culturing.

Well-defined thermosensitive poly[tri(ethylene glycol) monoethyl ether methacrylate] (P(TEGMA-EE)) brushes were synthesized on a solid substrate by the surface-initiated atom transfer radical polymerization of TEGMA-EE. The polymerization reaction was initiated by 2-bromo-2-methylpropionate groups immobilized on the surface of the wafers. The changes in the surface composition, morphology, philicity, and thickness that occurred at each step of wafer functionalization confirmed that all surface modification procedures were successful. Both the successful modification of the surface and bonding of the P(TEGMA-EE) layer were confirmed by X-ray photoelectron spectroscopy (XPS) measurements. The thickness of the obtained P(TEGMA-EE) layers increased with increasing polymerization time. The increase of environmental temperature above the cloud point temperature of P(TEGMA-EE) caused the changes of surface philicity. A simultaneous decrease in the polymer layer thickness confirmed the thermosensitive properties of these P(TEGMA-EE) layers. The thermosensitive polymer surfaces obtained were evaluated for the growth and harvesting of human fibroblasts (basic skin cells). At 37 °C, seeded cells adhered to and spread well onto the P(TEGMA-EE)-coated surfaces. A confluent cell sheet was formed within 24 h of cell culture. Lowering the temperature to an optimal value of 17.5 °C (below the cloud point temperature of the polymer, TCP, in cell culture medium) led to the separation of the fibroblast sheet from the polymer layer. These promising results indicate that the surfaces produced may successfully be used as substrate for engineering of skin tissue, especially for delivering cell sheets in the treatment of burns and slow-healing wounds.