Substrates mimicking the blastocyst geometry revert pluripotent stem cell to naivety.

Naive pluripotent stem cells have the highest developmental potential but their in vivo existence in the blastocyst is transient. Here we report a blastocyst motif substrate for the in vitro reversion of mouse and human pluripotent stem cells to a naive state. The substrate features randomly varied microstructures, which we call motifs, mimicking the geometry of the blastocyst. Motifs representing mouse-blastocyst-scaled curvature ranging between 15 and 62 mm-1 were the most efficient in promoting reversion to naivety, as determined by time-resolved correlative analysis. In these substrates, apical constriction enhances E-cadherin/RAC1 signalling and activates the mechanosensitive nuclear transducer YAP, promoting the histone modification of pluripotency genes. This results in enhanced levels of pluripotency transcription factor NANOG, which persist even after cells are removed from the substrate. Pluripotent stem cells cultured in blastocyst motif substrates display a higher development potential in generating embryoid bodies and teratomas. These findings shed light on naivety-promoting substrate design and their large-scale implementation.

Polymeric sheet actuators with programmable bioinstructivity.

Stem cells are capable of sensing and processing environmental inputs, converting this information to output a specific cell lineage through signaling cascades. Despite the combinatorial nature of mechanical, thermal, and biochemical signals, these stimuli have typically been decoupled and applied independently, requiring continuous regulation by controlling units. We employ a programmable polymer actuator sheet to autonomously synchronize thermal and mechanical signals applied to mesenchymal stem cells (MSCs). Using a grid on its underside, the shape change of polymer sheet, as well as cell morphology, calcium (Ca2+) influx, and focal adhesion assembly, could be visualized and quantified. This paper gives compelling evidence that the temperature sensing and mechanosensing of MSCs are interconnected via intracellular Ca2+ Up-regulated Ca2+ levels lead to a remarkable alteration of histone H3K9 acetylation and activation of osteogenic related genes. The interplay of physical, thermal, and biochemical signaling was utilized to accelerate the cell differentiation toward osteogenic lineage. The approach of programmable bioinstructivity provides a fundamental principle for functional biomaterials exhibiting multifaceted stimuli on differentiation programs. Technological impact is expected in the tissue engineering of periosteum for treating bone defects.

On Demand Sequential Release of (Sub)Micron Particles Controlled by Size and Temperature.

Polymeric devices capable of releasing submicron particles (subMP) on demand are highly desirable for controlled release systems, sensors, and smart surfaces. Here, a temperature-memory polymer sheet with a programmable smooth surface served as matrix to embed and release polystyrene subMP controlled by particle size and temperature. subMPs embedding at 80 °C can be released sequentially according to their size (diameter D of 200 nm, 500 nm, 1 µm) when heated. The differences in their embedding extent are determined by the various subMPs sizes and result in their distinct release temperatures. Microparticles of the same size (D ≈ 1 µm) incorporated in films at different programming temperatures Tp (50, 65, and 80 °C) lead to a sequential release based on the temperature-memory effect. The change of apparent height over the film surface is quantified using atomic force microscopy and the realization of sequential release is proven by confocal laser scanning microscopy. The demonstration and quantification of on demand subMP release are of technological impact for assembly, particle sorting, and release technologies in microtechnology, catalysis, and controlled release.

Thiol-Thioester Exchange Reactions in Precursors Enable pH-Triggered Hydrogel Formation.

Bio-interactive hydrogel formation in situ requires sensory capabilities toward physiologically relevant stimuli. Here, we report on pH-controlled in situ hydrogel formation relying on latent cross-linkers, which transform from pH sensors to reactive molecules. In particular, thiopeptolide/thio-depsipeptides were capable of pH-sensitive thiol-thioester exchange reactions to yield α,ω-dithiols, which react with maleimide-functionalized multi-arm polyethylene glycol to polymer networks. Their water solubility and diffusibility qualify thiol/thioester-containing peptide mimetics as sensory precursors to drive in situ localized hydrogel formation with potential applications in tissue regeneration such as treatment of inflamed tissues of the urinary tract.

Switching microobjects from low to high aspect ratios using a shape-memory effect.

Spherical particles from shape-memory polymers (SMP) can be stretched to ellipsoids with high aspect ratio (AR) and temporarily stabilized. They can switch back to low AR upon thermal stimulation. Here, the creation of an alternative shape-switching capability of particles from low to high AR is introduced, where a SMP matrix from polyvinyl alcohol (PVA) is used to create crosslinked high AR particles and to program the embedded micrometer-sized particles from a second SMP (oligo(ε-caprolactone) micronetworks, MN) with a low switching temperature Tsw. This programming proceeds through shape-recovery of the PVA matrix, from which the MN are harvested by PVA matrix dissolution. The use of a dissolvable SMP matrix may be a general strategy to efficiently create systems with complex moving capabilities.

Designing Cardiovascular Implants Taking in View the Endothelial Basement Membrane.

Insufficient endothelialization of cardiovascular grafts is a major hurdle in vascular surgery and regenerative medicine, bearing a risk for early graft thrombosis. Neither of the numerous strategies pursued to solve these problems were conclusive. Endothelialization is regulated by the endothelial basement membrane (EBM), a highly specialized part of the vascular extracellular matrix. Thus, a detailed understanding of the structure-function interrelations of the EBM components is fundamental for designing biomimetic materials aiming to mimic EBM functions. In this review, a detailed description of the structure and functions of the EBM are provided, including the luminal and abluminal interactions with adjacent cell types, such as vascular smooth muscle cells. Moreover, in vivo as well as in vitro strategies to build or renew EBM are summarized and critically discussed. The spectrum of methods includes vessel decellularization and implant biofunctionalization strategies as well as tissue engineering-based approaches and bioprinting. Finally, the limitations of these methods are highlighted, and future directions are suggested to help improve future design strategies for EBM-inspired materials in the cardiovascular field.

Venous and Arterial Endothelial Cells from Human Umbilical Cords: Potential Cell Sources for Cardiovascular Research.

Although cardiovascular devices are mostly implanted in arteries or to replace arteries, in vitro studies on implant endothelialization are commonly performed with human umbilical cord-derived venous endothelial cells (HUVEC). In light of considerable differences, both morphologically and functionally, between arterial and venous endothelial cells, we here compare HUVEC and human umbilical cord-derived arterial endothelial cells (HUAEC) regarding their equivalence as an endothelial cell in vitro model for cardiovascular research. No differences were found in either for the tested parameters. The metabolic activity and lactate dehydrogenase, an indicator for the membrane integrity, slightly decreased over seven days of cultivation upon normalization to the cell number. The amount of secreted nitrite and nitrate, as well as prostacyclin per cell, also decreased slightly over time. Thromboxane B2 was secreted in constant amounts per cell at all time points. The Von Willebrand factor remained mainly intracellularly up to seven days of cultivation. In contrast, collagen and laminin were secreted into the extracellular space with increasing cell density. Based on these results one might argue that both cell types are equally suited for cardiovascular research. However, future studies should investigate further cell functionalities, and whether arterial endothelial cells from implantation-relevant areas, such as coronary arteries in the heart, are superior to umbilical cord-derived endothelial cells.

Effect of Endothelial Culture Medium Composition on Platelet Responses to Polymeric Biomaterials.

Near-physiological in vitro thrombogenicity test systems for the evaluation of blood-contacting endothelialized biomaterials requires co-cultivation with platelets (PLT). However, the addition of PLT has led to unphysiological endothelial cell (EC) detachment in such in vitro systems. A possible cause for this phenomenon may be PLT activation triggered by the applied endothelial cell medium, which typically consists of basal medium (BM) and nine different supplements. To verify this hypothesis, the influence of BM and its supplements was systematically analyzed regarding PLT responses. For this, human platelet rich plasma (PRP) was mixed with BM, BM containing one of nine supplements, or with BM containing all supplements together. PLT adherence analysis was carried out in six-channel slides with plasma-treated cyclic olefin copolymer (COC) and poly(tetrafluoro ethylene) (PTFE, as a positive control) substrates as part of the six-channel slides in the absence of EC and under static conditions. PLT activation and aggregation were analyzed using light transmission aggregometry and flow cytometry (CD62P). Medium supplements had no effect on PLT activation and aggregation. In contrast, supplements differentially affected PLT adherence, however, in a polymer- and donor-dependent manner. Thus, the use of standard endothelial growth medium (BM + all supplements) maintains functionality of PLT under EC compatible conditions without masking the differences of PLT adherence on different polymeric substrates. These findings are important prerequisites for the establishment of a near-physiological in vitro thrombogenicity test system assessing polymer-based cardiovascular implant materials in contact with EC and PLT.

Potential Effects of Nonadherent on Adherent Human Umbilical Venous Endothelial Cells in Cell Culture.

The adherence and shear-resistance of human umbilical venous endothelial cells (HUVEC) on polymers is determined in vitro in order to qualify cardiovascular implant materials. In these tests, variable fractions of HUVEC do not adhere to the material but remain suspended in the culture medium. Nonadherent HUVEC usually stop growing, rapidly lose their viability and can release mediators able to influence the growth and function of the adherent HUVEC. The aim of this study was the investigation of the time dependent behaviour of HUVEC under controlled nonadherent conditions, in order to gain insights into potential influences of these cells on their surrounding environment in particular adherent HUVEC in the context of in vitro biofunctionality assessment of cardiovascular implant materials. Data from adherent or nonadherent HUVEC growing on polystyrene-based cell adhesive tissue culture plates (TCP) or nonadhesive low attachment plates (LAP) allow to calculate the number of mediators released into the culture medium either from adherent or nonadherent cells. Thus, the source of the inflammatory mediators can be identified. For nonadherent HUVEC, a time-dependent aggregation without further proliferation was observed. The rate of apoptotic/dead HUVEC progressively increased over 90% within two days. Concomitant with distinct blebbing and loss of membrane integrity over time, augmented releases of prostacyclin (PGI2, up to 2.91 ± 0.62 fg/cell) and platelet-derived growth factor BB (PDGF-BB, up to 1.46 ± 0.42 fg/cell) were detected. The study revealed that nonadherent, dying HUVEC released mediators, which can influence the surrounding microenvironment and thereby the results of in vitro biofunctionality assessment of cardiovascular implant materials. Neglecting nonadherent HUVEC bears the risk for under- or overestimation of the materials endothelialization potential, which could lead to the loss of relevant candidates or to uncertainty with regard to their suitability for cardiac applications. One approach to minimize the influence from nonadherent endothelial cells could be their removal shortly after observing initial cell adhesion. However, this would require an individual adaptation of the study design, depending on the properties of the biomaterial used.

Thermally-Induced Shape-Memory Behavior of Degradable Gelatin-Based Networks.

Shape-memory hydrogels (SMH) are multifunctional, actively-moving polymers of interest in biomedicine. In loosely crosslinked polymer networks, gelatin chains may form triple helices, which can act as temporary net points in SMH, depending on the presence of salts. Here, we show programming and initiation of the shape-memory effect of such networks based on a thermomechanical process compatible with the physiological environment. The SMH were synthesized by reaction of glycidylmethacrylated gelatin with oligo(ethylene glycol) (OEG) α,ω-dithiols of varying crosslinker length and amount. Triple helicalization of gelatin chains is shown directly by wide-angle X-ray scattering and indirectly via the mechanical behavior at different temperatures. The ability to form triple helices increased with the molar mass of the crosslinker. Hydrogels had storage moduli of 0.27-23 kPa and Young's moduli of 215-360 kPa at 4 °C. The hydrogels were hydrolytically degradable, with full degradation to water-soluble products within one week at 37 °C and pH = 7.4. A thermally-induced shape-memory effect is demonstrated in bending as well as in compression tests, in which shape recovery with excellent shape-recovery rates Rr close to 100% were observed. In the future, the material presented here could be applied, e.g., as self-anchoring devices mechanically resembling the extracellular matrix.

Unraveling the Interplay between Abiotic Hydrolytic Degradation and Crystallization of Bacterial Polyesters Comprising Short and Medium Side-Chain-Length Polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) have attracted attention as degradable (co)polyesters which can be produced by microorganisms with variations in the side chain. This structural variation influences not only the thermomechanical properties of the material but also its degradation behavior. Here, we used Langmuir monolayers at the air-water (A-W) interface as suitable models for evaluating the abiotic degradation of two PHAs with different side-chain lengths and crystallinity. By controlling the polymer state (semicrystalline, amorphous), the packing density, the pH, and the degradation mechanism, we could draw several significant conclusions. (i) The maximum degree of crystallinity for a PHA film to be efficiently degraded up to pH = 12.3 is 40%. (ii) PHA made of repeating units with shorter side-chain length are more easily hydrolyzed under alkaline conditions. The efficiency of alkaline hydrolysis decreased by about 65% when the polymer was 40% crystalline. (iii) In PHA films with a relatively high initial crystallinity, abiotic degradation initiated a chemi-crystallization phenomenon, detected as an increase in the storage modulus (E'). This could translate into an increase in brittleness and reduction in the material degradability. Finally, we demonstrate the stability of the measurement system for long-term experiments, which allows degradation conditions for polymers that could closely simulate real-time degradation.

Controlling Actuation Performance in Physically Cross-Linked Polylactone Blends Using Polylactide Stereocomplexation.

Within the field of shape-changing materials, synthetic chemical modification has been widely used to introduce key structural units and subsequently expand the mechanical functionality of actuator devices. The introduction of architectural elements that facilitate in situ control over mechanical properties and complete geometric reconfiguration of a device is highly desirable to increase the morphological diversity of polymeric actuator materials. The subject of the present study is a multiblock copolymer with semicrystalline poly(l-lactide) and poly(ε-caprolactone) (PLLA-PCL) segments. By harnessing the stereocomplexation of copolymer chains with a poly(d-lactide) oligomer (PDLA), we provide anchoring points for physical network formation and demonstrate how a blending process can be used to efficiently vary the mechanical properties of a shape-memory actuator. We investigate the effect of molecular structure on the actuation performance of the material in cyclic thermomechanical tests, with a maximum reversible shape change εrev' = 13.4 ± 1.5% measured at 3.1 wt % of polylactide stereocomplex content in the multiblock copolymer matrix. The thermophysical properties, crystalline structure, and phase morphology were analyzed by DSC, WAXS and AFM respectively, elucidating the structure-to-function relationship in physically cross-linked blended materials. The work demonstrates a one-step technique for manufacturing a polymeric actuator and tuning its performance in situ. This approach should greatly improve the efficiency of physically cross-linked actuator fabrication, allowing composition and physical behavior to be precisely and easily controlled.

Shape-Memory Effect by Sequential Coupling of Functions over Different Length Scales in an Architectured Hydrogel.

The integration of functions in materials in order to gain macroscopic effects in response to environmental changes is an ongoing challenge in material science. Here, functions on different hierarchical levels are sequentially linked to translate a pH-triggered conformational transition from the molecular to the macroscopic level to induce directed movements in hydrogels. When the pH is increased, lysine-rich peptide molecules change their conformation into a ß-hairpin structure because of the reduced electrostatic repulsion among the deprotonated amino groups. Coupled to this conformation change is the capability of the ß-hairpin motifs to subsequently assemble into aggregates acting as reversible cross-links, which are used as controlling units to fix a temporary macroscopic shape. A structural function implemented into the hydrogel by a microporous architecture-enabled nondisruptive deformation upon compression by buckling of pore walls and their elastic recovery. Coupled to this structural function is the capability of the porous material to enhance the diffusion of ions into the hydrogel and to keep the dimension of the macroscopic systems almost constant when the additional cross-links are formed or cleaved as it limits the dimensional change of the pore walls. Covalent cross-linking of the hydrogel into a polymer network acted as gear shift to ensure translation of the function on the molecular level to the macroscopic dimension. In this way, the information of a directed shape-shift can be programmed into the material by mechanical deformation and pH-dependent formation of temporary net points. The information could be read out by lowering the pH. The peptides reverted back into their original random coil conformation and the porous polymer network could recover from the previously applied elastic deformation. The level of multifunctionality of the hydrogels can be increased by implementation of additional orthogonal functions such as antimicrobicity by proper selection of multifunctional peptides, which could enable sophisticated biomedical devices.

Salt-Induced Shape-Memory Effect in Gelatin-Based Hydrogels.

Hydrophilic biopolymers display a strong tendency for self-organization into stable secondary, tertiary, and quaternary structures in aqueous environments. These structures are sensitive to changes in external conditions, such as temperature, pH or ions/salts, which may lead to molecular and/or macroscopic transitions. Here, we report on biopolymer-based stimuli-sensitive switchable matrices showing a shape-memory function as an output being alternatively switched by two different input signals, such as environmental changes in salt concentration or temperature. This was realized by implementing a shape-memory function in hydrogels based on the coil-to-helix transition of protein chains in gelatin-based networks. The hydrogels exhibited mechanical properties similar to that of soft tissue (storage modulus G' = 1-100 kPa) and high swelling capabilities (Q = 1000-3000 vol %). In these gelatin-based networks, the covalent netpoints defined the permanent shape while after deformation helicalization of the gelatin acted as reversible stimuli-sensitive switches providing additional crosslinks capable of fixing the deformed temporary shape. By using either chaotropic salts to suppress gelatin helicalization or kosmotropic salts to support conformational changes of gelatin toward a helical orientation, these additional crosslinks could be cleaved or formed. In bending experiments, the strain fixity (Rf) and strain recovery ratios (Rr) were determined. While Rf ranged from 65 to 95% and was depending on the network composition, Rr were independent of the hydrogel composition with values about 100%. In addition, Rf and Rr were independent of the type of chaotropic salt that was used in this study, showing equal Rf and Rr values for MgCl2, NaSCN, and Mg(SCN)2.

Matching Magnetic Heating and Thermal Actuation for Sequential Coupling in Hybrid Composites by Design.

Sequentially coupling two material functions requires matching the output from the first with the input of the second function. Here, magnetic heating controls thermal actuation of a hybrid composite in a challenging system environment causing an elevated level of heat loss. The concept is a hierarchical design consisting of an inner actuator of nanocomposite material, which can be remotely heated by exposure to an alternating magnetic field (AMF) and outer layers of a porous composite system with a closed pore morphology. These porous layers act as heat insulators and as barriers to the surrounding water. By exposure to the AMF, a local bulk temperature of 71 °C enables the magnetic actuation of the device, while the temperature of the surrounding water is kept below 50 °C. Interestingly, the heat loss during magnetic heating leads to an increase of the water phase (small volume) temperature. The temperature increase is able to sequentially trigger an adjacent thermal actuator attached to the actuator composite. In this way it could be demonstrated how the AMF is able to initiate two kinds of independent actuations, which might be interesting for robotics operating in aqueous environments.

Shape-Programmable Architectured Hydrogels Sensitive to Ultrasound.

On-demand motion of highly swollen polymer systems can be triggered by changes in pH, ion concentrations, or by heat. Here, shape-programmable, architectured hydrogels are introduced, which respond to ultrasonic-cavitation-based mechanical forces (CMF) by directed macroscopic movements. The concept is the implementation and sequential coupling of multiple functions (swellability in water, sensitivity to ultrasound, shape programmability, and shape-memory) in a semi-interpenetrating polymer network (s-IPN). The semi-IPN-based hydrogels are designed to function through rhodium coordination (Rh-s-IPNH). These coordination bonds act as temporary crosslinks. The porous hydrogels with coordination bonds (degree of swelling from 300 ± 10 to 680 ± 60) exhibit tensile strength σmax up to 250 ± 60 kPa. Shape fixity ratios up to 90% and shape recovery ratios up to 94% are reached. Potential applications are switches or mechanosensors.

Bioperspectives for Shape-Memory Polymers as Shape Programmable, Active Materials.

Within the natural world, organisms use information stored in their material structure to generate a physical response to a wide variety of environmental changes. The ability to program synthetic materials to intrinsically respond to environmental changes in a similar manner has the potential to revolutionize material science. By designing polymeric devices capable of responsively changing shape or behavior based on information encoded into their structure, we can create functional physical behavior, including a shape-memory and an actuation capability. Here we highlight the stimuli-responsiveness and shape-changing ability of biological materials and biopolymer-based materials, plus their potential biomedical application, providing a bioperspective on shape-memory materials. We address strategies to incorporate a shape-memory (actuation) function in polymeric materials, conceptualized in terms of its relationship with inputs (environmental stimuli) and outputs (shape change). Challenges and opportunities associated with the integration of several functions in a single material body to achieve multifunctionality are discussed. Finally, we describe how elements that sense, convert, and transmit stimuli have been used to create multisensitive materials.

Molecular Insights into the Physical Adsorption of Amphiphilic Protein PhaF onto Copolyester Surfaces.

Phasins are amphiphilic proteins located at the polymer-cytoplasm interface of bacterial polyhydroxyalkanoates (PHA). The immobilization of phasins on biomaterial surfaces is a promising way to enhance the hydrophilicity and supply cell-directing elements in bioinstructing processes. Optimizing the physical adsorption of phasins requires deep insights into molecular processes during polymer-protein interactions to preserve their structural conformation while optimizing surface coverage. Here, the assembly, organization, and stability of phasin PhaF from Pseudomonas putida at interfaces is disclosed. The Langmuir technique, combined with in situ microscopy and spectroscopic methods, revealed that PhaF forms stable and robust monolayers at different temperatures, with an almost flat orientation of its α-helix at the air-water interface. PhaF adsorption onto preformed monolayers of poly[(3-R-hydroxyoctanoate)-co-(3-R-hydroxyhexanoate)] (PHOHHx), yields stable mixed layers below π = ∼15.7 mN/m. Further insertion induces a molecular reorganization. PHOHHx with strong surface hydrophobicity is a more adequate substrate for PhaF adsorption than the less hydrophobic poly[(rac-lactide)-co-glycolide] (PLGA). The observed orientation of the main axis of the protein in relation to copolyester interfaces ensures the best exposure of the hydrophobic residues, providing a suitable coating strategy for polymer functionalization.

Langmuir Monolayers as Tools to Study Biodegradable Polymer Implant Materials.

Langmuir monolayers provide a fast and elegant route to analyze the degradation behavior of biodegradable polymer materials. In contrast to bulk materials, diffusive transport of reactants and reaction products in the (partially degraded) material can be neglected at the air-water interface, allowing for the study of molecular degradation kinetics in experiments taking less than a day and in some cases just a few minutes, in contrast to experiments with bulk materials that can take years. Several aspects of the biodegradation behavior of polymer materials, such as the interaction with biomolecules and degradation products, are directly observable. Expanding the technique with surface-sensitive instrumental techniques enables evaluating the evolution of the morphology, chemical composition, and the mechanical properties of the degrading material in situ. The potential of the Langmuir monolayer degradation technique as a predictive tool for implant degradation when combined with computational methods is outlined, and related open questions and strategies to overcome these challenges are pointed out.

Revival of transcatheter PFO closure: A meta-analysis of randomized controlled trials - impact of shunt size and age.

BACKGROUND: Transcatheter foramen ovale closure (TPC) has emerged as a potential treatment option for patients with cryptogenic strokes and persistent foramen ovale (PFO). However, previous randomized controlled trials could hardly demonstrate any benefit compared to medical treatment (Med-Tx). Recently new data have become available which may change current practice of transcatheter PFO closure. METHODS: A systematic review and meta-analysis comparing TPC and Med-Tx based on all available multicentric randomized controlled trials was performed. The primary outcome of interest was the recurrence of stroke in both groups. RESULTS: Five studies met the inclusion criteria with 1829 patients in the TPC and 1622 in the Med-Tx group. The median follow-up was 4 years. In the intention-to-treat analysis we found a statistically significant relative risk reduction in recurrence of strokes in the TPC group compared to the Med-Tx group (pooled hazard ratio (HR): 0.32; 95% CI: 0.13-0.8; P = .018). Excluding one study due to potential publication bias resulted in a pooled HR of 0.48 (95% CI: 0.25-0.91, P = .024). Patients younger than 45 years of age (pooled HR: 0.35; 95% CI: 0.16-0.75; P = .007) and those with moderate to severe shunt (pooled HR: 0.28; 95% CI: 0.14-0.55; P < .001) were more likely to benefit from closure. CONCLUSION: According to our meta-analysis TPC plus antiplatelets was superior in terms of stroke prevention when compared to Med-Tx. Furthermore, patients with moderate to severe shunts and those younger than 45 years of age were found to benefit most from TPC.