Phase transition and potential biomedical applications of thermoresponsive compositions based on polysaccharides, proteins and DNA: A review.

Smart thermoresponsive polymers have long attracted attention as materials of a great potential for biomedical applications, mainly for drug delivery, tissue engineering and wound dressing, with a special interest to injectable hydrogels. Poly-N-isopropylacrylamide (PNIPAM) is the most important synthetic thermoresponsive polymer due to its physiologically relevant transition temperature. However, the use of unmodified PNIPAM encounters such problems as low biodegradability, low drug loading capacity, slow response to thermal stimuli, and insufficient mechanical robustness. The use of natural polysaccharides and proteins in combinations with PNIPAM, in the form of grafted copolymers, IPNs, microgels and physical mixtures, is aimed at overcoming these drawbacks and creating dual-functional materials with both synthetic and natural polymers' properties. When developing such compositions, special attention should be paid to preserving their key property, thermoresponsiveness. Addition of hydrophobic and hydrophilic fragments to PNIPAM is known to affect its transition temperature. This review covers various classes of natural polymers - polysaccharides, fibrous and non-fibrous proteins, DNA - used in combination with PNIPAM for the prospective biomedical purposes, with a focus on their phase transition temperatures and its relation to the natural polymer's structure.

Macromolecular crowding transforms regenerative medicine by enabling the accelerated development of functional and truly three-dimensional cell assembled micro tissues.

Scaffold-free in vitro organogenesis exploits the innate ability of cells to synthesise and deposit their own extracellular matrix to fabricate tissue-like assemblies. Unfortunately, cell-assembled tissue engineered concepts require prolonged ex vivo culture periods of very high cell numbers for the development of a borderline three-dimensional implantable device, which are associated with phenotypic drift and high manufacturing costs, thus, hindering their clinical translation and commercialisation. Herein, we report the accelerated (10 days) development of a truly three-dimensional (338.1 ± 42.9 µm) scaffold-free tissue equivalent that promotes fast wound healing and induces formation of neotissue composed of mature collagen fibres, using human adipose derived stem cells seeded at only 50,000 cells/cm2 on an poly (N-isopropylacrylamide-co-N-tert-butylacrylamide (PNIPAM86-NTBA14) temperature-responsive electrospun scaffold and grown under macromolecular crowding conditions (50 µg/ml carrageenan). Our data pave the path for a new era in scaffold-free regenerative medicine.

Thin Thermoresponsive Polymer Films for Cell Culture: Elucidating an Unexpected Thermal Phase Behavior by Atomic Force Microscopy.

Application of poly-N-isopropylacrylamide (PNIPAM) and its more hydrophobic copolymers with N-tert-butylacrylamide (NtBA) as supports for cell sheets has been validated in numerous studies. The binary systems of these polymers with water are characterized by a lower critical solution temperature (LCST) in a physiologically favorable region. Upon lowering the temperature below the LCST, PNIPAM chains undergo a globule-to-coil transition, causing the film dissolution and cell sheet detachment. The character of the PNIPAM-water miscibility behavior is rather complex and not completely understood. Here, we applied atomic force microscopy to track the phase transition in thin films of linear thermoresponsive (co)polymers (PNIPAM and PNIPAM-co-NtBA) prepared by spin-coating. We studied the films' Young's modulus, roughness, and thickness in air and in distilled water in a full thermal cycle. In dry films, in the absence of water, all the measured parameters remained invariant. The swollen films in water above the LCST were softer by 2-3 orders of magnitude and about 10 times rougher than the corresponding dry films. Upon lowering the temperature to the LCST, the films passed through the phase transition observed as a drastic drop of Young's modulus (about an order of magnitude) and decrease in roughness in both polymers in a narrow temperature range. However, the films did not lose their integrity and demonstrated almost fully reversible changes in the mechanical properties and roughness. The thermal dependence of the films' thickness confirmed that they dissolved only partially and required an external force to induce the complete destruction. The reversible thermal behavior which is generally not expected from non-cross-linked polymers is a key finding, especially with respect to their practical application in cell culture. Both the thermodynamic and kinetic factors, as well as the confinement effect, may be responsible for this peculiar film robustness, which requires overcooling and the aid of an external force to destroy the film.

Fabrication of hydrogel scaffolds via photocrosslinking of methacrylated silk fibroin.

Silk fibroin is a promising biomaterial for tissue engineering due to its valuable mechanical and biological properties. However, being a natural product and a protein, it lacks the processability and uniform quality of an advanced synthetic material. Here we propose a way to overcome this contradiction using novel fibroin photocrosslinkable derivative (FBMA). FBMA was synthesized by methacrylation of native fibroin nucleophilic side groups. It was dissolved in either formic acid (FA) or hexafluoroisopropanol (HFIP), and the obtained solutions were photocrosslinked into hydrogel scaffolds of various structural forms including films, micropatterns, pads and macroporous sponges. UV-exposition of dry FBMA films through a photomask created complex microscaled patterns of the polymer. The nature of the solvent affected the properties of resulting hydrogels. When HFIP was used as the solvent, the resulting hydrogels had a storage modulus ∼4 times higher than that of hydrogels fabricated using FA and ∼20 times higher compared to the reference hydrogel obtained from pristine fibroin. Both FBMA-based hydrogels were biocompatible and supported fibroblast adhesion and growth in vitro. Cells cultivated on FBMA scaffolds produced with HFIP exhibited more spread phenotype at 4 and 24 h of cultivation, consistent with increased stiffness of the hydrogel. Hence, FBMA is an attractive material for fabrication of micropatterned scaffolds of centimeter-scale size with minutely tunable physico-chemical properties via convenient and reproducible technological processes, applicable for rapid prototyping.

Film Thickness Determines Cell Growth and Cell Sheet Detachment from Spin-Coated Poly(N-Isopropylacrylamide) Substrates.

Poly(N-isopropylacrylamide) (pNIPAm) is widely used to fabricate thermoresponsive surfaces for cell sheet detachment. Many complex and expensive techniques have been employed to produce pNIPAm substrates for cell culture. The spin-coating technique allows rapid fabrication of pNIPAm substrates with high reproducibility and uniformity. In this study, the dynamics of cell attachment, proliferation, and function on non-cross-linked spin-coated pNIPAm films of different thicknesses were investigated. The measurements of advancing contact angle revealed increasing contact angles with increasing film thickness. Results suggest that more hydrophilic 50 and 80 nm thin pNIPAm films are more preferable for cell sheet fabrication, whereas more hydrophobic 300 and 900 nm thick spin-coated pNIPAm films impede cell attachment. These changes in cell behavior were correlated with changes in thickness and hydration of pNIPAm films. The control of pNIPAm film thickness using the spin-coating technique offers an effective tool for cell sheet-based tissue engineering.

Novel biodegradable star-shaped polylactide scaffolds for bone regeneration fabricated by two-photon polymerization.

AIM: To assess the properties of 3D biodegradable scaffolds fabricated from novel star-shaped poly(D,L-lactide) (SSL) materials for bone tissue regeneration. MATERIALS & METHODS: The SSL polymer was synthesized using an optimized synthetic procedure and applied for scaffold fabrication by the two-photon polymerization technique. The osteogenic differentiation was controlled using human adipose-derived stem cells cultured for 28 days. The SSL scaffolds with or without murine MSCs were implanted into the cranial bone of C57/Bl6 mice. RESULTS: The SSL scaffolds supported differentiation of human adipose-derived stem cells toward the osteogenic lineage in vitro. The SSL scaffolds with murine MSCs enhanced the mineralized tissue formation. CONCLUSION: The SSL scaffolds provide a beneficial microenvironment for the osteogenic MSCs' differentiation in vitro and support de novo bone formation in vivo.

Controlling cellular activity by manipulating silicone surface roughness.

Silicone elastomers exhibit a broad range of beneficial properties that are exploited in biomaterials. In some cases, however, problems can arise at silicone elastomer interfaces. With breast implants, for example, the fibrous capsule that forms at the silicone interface can undergo contracture, which can lead to the need for revision surgery. The relationship between surface topography and wound healing--which could impact on the degree of contracture--has not been examined in detail. To address this, we prepared silicone elastomer samples with rms surface roughnesses varying from 88 to 650 nm and examined the growth of 3T3 fibroblasts on these surfaces. The PicoGreen assay demonstrated that fibroblast growth decreased with increases in surface roughness. Relatively smooth (approximately 88 nm) PDMS samples had ca. twice as much fibroblast DNA per unit area than the 'bumpy' (approximately 378 nm) and very rough (approximately 604 and approximately 650 nm) PDMS samples. While the PDMS sample with roughness of approximately 650 nm had significantly fewer fibroblasts at 24h than the TCP control, fibroblasts on the smooth silicone surprisingly reached confluence much more rapidly than on TCP, the gold standard for cell culture. Thus, increasing the surface roughness at the sub-micron scale could be a strategy worthy of consideration to help mitigate fibroblast growth and control fibrous capsule formation on silicone elastomer implants.

The effects of vinblastine on smooth muscle cells in vitro: evaluation of a therapeutic window for the treatment of restenosis.

The development of drug-eluting stents (DES) to combat the problem of in-stent restenosis has revolutionized interventional cardiology. However, concerns have emerged about the risk of late angiographic stent thromboses associated with DES. The evaluation and width of the therapeutic window of a particular DES system is of huge importance to its safety and efficacy. In this study, the effects of vinblastine, an antimitotic drug, on smooth muscle cells in vitro is analyzed. The change in levels of proliferation, activity, migration, and viability in human coronary artery smooth muscle cells was measured at a range of concentrations and over a number of time points. These findings were then compared with those of a previous study on the effects of vinblastine on endothelial cells, and an optimum working concentration range was evaluated. This study suggests that the concentration of vinblastine most appropriate in restenosis treatment would be between 0.1 and 1 nM. At this concentration, vinblastine exerts a distinct effect on smooth muscle cell proliferation without detrimental effects on endothelial cell viability. It was also found that vinblastine affects certain cellular activities such as migration in a threshold-independent manner, suggesting that very low doses could be active against the processes of restenosis.

Polarity assessment of thermoresponsive poly(NIPAM-co-NtBA) copolymer films using fluorescence methods.

The in-situ, non-contact, and non-destructive measurement of the physicochemical properties such as the polarity of thin, hydrophilic polymer films is desirable in many areas of polymer science. Polarity is a complex factor and encompasses a range of non-covalent interactions including dipolarity/polarizability and hydrogen bonding. A polarity measurement method based on fluorescence would be ideal, but the key challenge is to identify suitable probes which can accurately measure specific polarity related parameters. In this manuscript we assess a variety of fluorophores for measuring the polarity of a series of relatively hydrophilic, thermoresponsive N-isopropylacrylamide/N-tert-butylacrylamide (NIPAM/NtBA) copolymers. The emission properties of both pyrene and 3-Hydroxyflavone (3-HF) based fluorophores were measured in dry polymer films. In the case of pyrene, a relatively weak, linear relationship between polymer composition and the ratio of the first to the third vibronic band of the emission spectrum (I(1)/I(3)) is observed, but pyrene emission is very sensitive to temperature and thus not suitable for robust polarity measurements. The 3-HF fluorophores which can undergo an excited-state intramolecular proton transfer (ESIPT) reaction have a dual band fluorescence emission that exhibits strong solvatochromism. Here we used 4'-diethylamino-3-hydroxyflavone (FE), 5,6-benzo-4'-diethylamino-3-hydroxyflavone (BFE), and 4 -diethylamino-3-hydroxy-7-methoxyflavone (MFE). The log ratio of the dual band fluorescence emission (log (I(N*)/I(T*))) of 3-HF doped, dry, NIPAM-NtBA copolymer films were found to depend linearly on copolymer composition, with increasing hydrophobicity (greater NtBA fraction) leading to a decrease in the value of log (I(N*)/I(T*)). However, the ESIPT process in the polymer matrix was found to be irreversible, non-equilibrated and occurs over a much longer timescale in comparison to the results previously reported for liquid solvents.

Measuring the micro-polarity and hydrogen-bond donor/acceptor ability of thermoresponsive N-isopropylacrylamide/N-tert-butylacrylamide copolymer films using solvatochromic indicators.

Thin polymer films are important in many areas of biomaterials research, biomedical devices, and biological sensors. The accurate in situ measurement of multiple physicochemical properties of thin polymer films is critical in understanding biocompatibility, polymer function, and performance. In this work we demonstrate a facile spectroscopic methodology for accurately measuring the micro-polarity and hydrogen-bond donor/acceptor ability for a series of relatively hydrophilic thermoresponsive copolymers. The micro-polarity of the N-isopropylacrylamide (NIPAM) and N-tert-butylacrylamide (NtBA) co-polymers was evaluated by means of the E(T)(30), alpha, beta, and pi empirical solvatochromic polarity parameters. The data shows that increasing the NtBA fraction in the dry copolymer film reduces polarity and hydrogen-bonding ability. Within the Kamlet-Taft polarity framework, the NIPAM/NtBA copolymer films are strong hydrogen-bond acceptors, strongly dipolar/polarizable, and rather moderate hydrogen-bond donors. This characterization provides a more comprehensive physicochemical description of polymers, which aids the interpretation of film performance. Comparison of the measured E(T)(30) values with literature data for other water-soluble polymers show that dry NIPAM/NtBA copolymers are slightly more polar than poly(ethylene oxide), less polar than polyvinylalcohol, and approximately the same polarity as poly(N-vinyl-2-pyrrolidone). These findings indicate that this spectroscopic method is a facile, rapid, and nondestructive methodology for measuring polymer properties in situ, suitable for most biomaterials research laboratories.

Human coronary artery smooth muscle cell response to a novel PLA textile/fibrin gel composite scaffold.

Previous studies have demonstrated the potential of fibrin as a cell carrier for cardiovascular tissue engineering applications. Unfortunately, fibrin exhibits poor mechanical properties. One method of addressing this issue is to incorporate a textile in fibrin to provide structural support. However, it is first necessary to develop a deeper understanding of the effect of the textile on cell response. In this study, the cytotoxicity of a polylactic acid (PLA) warp-knit textile was assessed with human coronary artery smooth muscle cells (HCASMC). Subsequently, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was employed to examine the gene expression of HCASMC embedded in fibrin with and without the textile. Five genes were examined over a 3-week period: smooth muscle alpha-actin (SMalphaA), myosin heavy chain 11 smooth muscle (SM1/SM2), calponin, myosin heavy chain 10 non-muscle (SMemb) and collagen. Additionally, a microarray analysis was performed to examine a wider range of genes. The knitting process did not adversely affect the cell response; there was no dramatic change in cell number or metabolic rate compared to the negative control. After 3 weeks, there was no significant difference in gene expression, except for a slight decrease of 10% in SMemb in the fibrin with textile. After 3 weeks, there were no obvious cytotoxic effects observed as a result of the knitting process and the gene expression profile did not appear to be altered in the presence of the mesh in the fibrin gel.

Estimation of the strength of adhesion between a thermoresponsive polymer coating and nitinol wire.

As polymer coatings become more widely used in the biomedical device industry, both to improve biocompatibility and as coatings for localised drug delivery, quantitative methods to measure the adhesive strength between coatings and substrates become a very important consideration. The aim of this study was to take a method for estimating the interfacial fracture toughness of a film to a flat substrate and apply it to Nitinol wires used in the production of medical devices. An investigation into the affect of surface roughness on the fracture toughness was also conducted. For the present study, a thermoresponsive based Poly (N-isopropylacrylamide) polymer was coated onto nitinol wire substrates and the adhesion strength between the polymer and wire was measured using a nanoindentation technique. Different surface treated nitinol wires, with different surface topography and roughness were used, and the affect of these surface properties on adhesion strength was investigated. Results showed that it was possible to apply the delamination technique to wire samples and obtain fracture toughness values. Results also showed that the surface roughness is an important parameter that can affect the adhesion between a coating and the substrate. It was found that, as the average surface roughness increased so also did the adhesive strength between the coating and wire sample.

Intact endothelial cell sheet harvesting from thermoresponsive surfaces coated with cell adhesion promoters.

Recently, with the development of smart polymers, research has looked to using thermoresponsive polymers as cell culture substrates. These novel surfaces allow the cultivation of cells without enzymes using the thermoresponsive phase transition property of poly(N-isopropylacrylamide) (PNIPAAm). However, this requires expensive techniques to generate a sufficiently thin film that allows cell adhesion. In this study, we looked at simple solvent cast films which normally show poor cell adhesion, but here the films are coated with cell adhesion promoters (CAPs) to improve cell growth without altering the copolymer thermoresponsive behaviour.A copolymer of PNIPAAm and N-tert-butylacrylamide (NtBAm) with a ratio of 85:15, respectively, was synthesized and solvent cast. The copolymer films were coated with CAPs, such as collagen, fibronectin and laminin, to increase their cell adhesion and growth properties. Cell activity measured by the alamarBlue assay showed similar results for coated copolymer films and standard tissue culture plastic controls. Deposition of CAPs on to the copolymer films was characterized by scanning electron microscopy and atomic force microscopy. Cell detachment from the copolymer films is not affected by the surface coatings of CAPs, and endothelial cells are recovered as an intact sheet, which has great potential for uses in tissue engineering applications. The results demonstrate a versatile method for the cultivation of cells while eliminating the need for the use of digestive enzymes such as trypsin. This study shows that cultivation on physically bonded PNIPAAm copolymers is viable and achievable by relatively simple methods.

Cell growth and detachment from protein-coated PNIPAAm-based copolymers.

The cultivation of cells requires the use of unfavorable proteolytic enzymes, which cause cell-surface modification and also need considerable optimization. Recently, with the development of smart polymers, research has looked to using thermoresponsive polymers as cell culture substrates. These novel surfaces allow the cultivation of cells without using enzymes by utilizing the thermoresponsive phase transition property of poly(N-isopropylacrylamide) (PNIPAAm). Copolymers of PNIPAAm and N-tert-butyl-acrylamide (NtBAm) with varying ratios were synthesized and solvent cast. The copolymer films are coated with cell adhesion promoters such as collagen, poly-L-lysine, and laminin to increase their cell adhesion and growth properties. Cell activity measured by the alamarBlue and PicoGreen assays is similar for coated copolymer films and standard tissue culture plastic controls. Deposition of cell adhesion promoters onto the copolymer films was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cell detachment from the copolymer films is dependent on copolymer composition and is not affected by the surface coatings of extracellular matrix (ECM) proteins. The results demonstrate a versatile method for the cultivation of cells while eliminating the need for the use of digestive enzymes such as trypsin.

Thermoresponsive poly(N-isopropylacrylamide) copolymers: contact angles and surface energies of polymer films.

Surface properties of poly(N-isopropylacrylamide) (PNIPAM) copolymer films were studied by contact angle measurements and optical and atomic force microscopy. We prepared a series of copolymers of N-isopropylacrylamide with N-tert-butylacrylamide (NtBA) in order of increasing hydrophobicity. The measurements of the advancing contact angle of water at 37 degrees C were hampered by the observation of a distinct stick/slip pattern on all polymers in the series with the exception of poly(NtBA) (PNtBA). We attributed this behavior to the film deformation by the vertical component of liquid surface tension leading to the pinning of the moving contact line. This was confirmed by the observation of a ridge formed at the pinned contact line by optical microscopy. However, meaningful contact (without the stick/slip pattern and with a time-independent advancing contact angle) angles for this thermoresponsive polymer series could be obtained with carefully selected organic liquids. We used the Li and Neumann equation of state to calculate the surface energy and contact angles of water for all polymers in the series of copolymers and van Oss, Chaudhury, and Good (vOCG) acid-base theory for PNtBA. The surface energies of the thermoresponsive polymers were in the range of 38.9 mJ/m2 (PNIPAM) to 31 mJ/m2 (PNtBA) from the equation of state approach. The surface energy of PNtBA calculated using vOCG theory was 29.0 mJ/m2. The calculated contact angle for PNIPAM (74.5 +/- 0.2 degrees ) is compared with previously reported contact angles obtained for PNIPAM-modified surfaces.

Local drug delivery in restenosis injury: thermoresponsive co-polymers as potential drug delivery systems.

The success of percutaneous transluminal coronary angioplasty in treatment of acute coronary syndromes has been compromised by the incidence of restenosis. The physical insult of balloon insertion can damage or remove the endothelial monolayer, thereby generating a prothrombotic surface. The resulting inappropriate response to injury can also lead to penetration of inflammatory cells, conversion of the underlying media to a synthetic phenotype, deposition of extracellular matrix, constrictive remodeling, and neointimal hyperplasia. While stent implantation at the time of balloon insertion has offset some of these events, inflammatory responses to the implanted biomaterial (stent) and intimal hyperplasia are still prominent features of the procedure, leading in 20-30% of cases to in-stent restenosis within a year. Systemic delivery of drugs designed to offset in-stent restenosis injury has been largely unsuccessful, which has led to the development of strategies for coating stents with drugs for local delivery. Drug-eluting stents constitute an innovative means of further reducing the incidence of restenosis injury and clinical trials have shown encouraging results. This review focuses on properties of a class of environment-sensitive hydrogels, the N-isopropylacrylamide-based thermoresponsive co-polymers, on their potential roles as stent coatings, on their demonstrated ability to incorporate and release drugs that modify vascular endothelial and smooth muscle cell functions, and on issues that still await clarification, prior to their adoption in a clinical setting.

Interaction of soft condensed materials with living cells: phenotype/transcriptome correlations for the hydrophobic effect.

The assessment of biomaterial compatibility relies heavily on the analysis of macroscopic cellular responses to material interaction. However, new technologies have become available that permit a more profound understanding of the molecular basis of cell-biomaterial interaction. Here, both conventional phenotypic and contemporary transcriptomic (DNA microarray-based) analysis techniques were combined to examine the interaction of cells with a homologous series of copolymer films that subtly vary in terms of surface hydrophobicity. More specifically, we used differing combinations of N-isopropylacrylamide, which is presently used as an adaptive cell culture substrate, and the more hydrophobic, yet structurally similar, monomer N-tert-butylacrylamide. We show here that even discrete modifications with respect to the physiochemistry of soft amorphous materials can lead to significant impacts on the phenotype of interacting cells. Furthermore, we have elucidated putative links between phenotypic responses to cell-biomaterial interaction and global gene expression profile alterations. This case study indicates that high-throughput analysis of gene expression not only can greatly refine our knowledge of cell-biomaterial interaction, but also can yield novel biomarkers for potential use in biocompatibility assessment.

Poly(N-isopropylacrylamide) co-polymer films as potential vehicles for delivery of an antimitotic agent to vascular smooth muscle cells.

INTRODUCTION: Local delivery of antimitotic agents is a potential therapeutic strategy for protection of injured coronary vasculature against intimal hyperplasia and restenosis. This study sought to establish the principle that thermoresponsive poly(N-isopropylacrylamide) co-polymer films can be used to deliver, in a controlled manner, an antimitotic agent to vascular smooth muscle cells (VSMC). METHODS: A series of co-polymer films was prepared, using varying ratios (w/w) of N-isopropylacrylamide (NiPAAm) monomer to N-tert-butylacrylamide (NtBAAm) and loaded with the antimitotic agent colchicine (100 nmol/film) at room temperature. RESULTS: The extent of colchicine release at 37 degrees C was inversely proportional to the amount of NtBAAm in co-polymer films: release after 48 h from 85:15, 65:35 and 50:50 (NiPAAm:NtBAAm) films was 26, 17 and 0.5 nmol, respectively. In cytotoxicity studies, when medium incubated with co-polymers for 24 h (in the absence of colchicine) was further incubated with target bovine aortic smooth muscle cells (BASMC), no loss of cell viability occurred. Colchicine released from all three co-polymer films significantly inhibited proliferation and random migration of BASMC: 100 nM colchicine (released from 65:35 NiPAAm:NtBAAm) reduced cell proliferation to 25.7+/-1.7% of levels seen in the absence of colchicine (control) and random cell migration to 37.7+/-5.7% of control (mean+/-S.E.M., n = 3, P < .01 and P < .05, respectively). The magnitudes of these effects were comparable to those seen in separate experiments with native colchicine and were observed in samples of released colchicine which had been stored at -20 degrees C for up to 6 months. CONCLUSIONS: This study has shown that the release of the antimitotic agent colchicine, from NiPAAm/NtBAAm co-polymer films can be manipulated by changes in co-polymer composition. Furthermore, such drug released at 37 degrees C retains comparable bioactivity to that of native colchicine.