This study describes the development and cell culture application of nanometer thick photocrosslinkable thermoresponsive polymer films prepared by physical adsorption. Two thermoresponsive polymers, poly(N‐isopropylacrylamide (NIPAm)‐co‐acrylamidebenzophenone (AcBzPh)) and poly(NIPAm‐co‐AcBzPh‐co‐N‐tertbutylacrylamide) are investigated. Films are prepared both above and below the polymers' lower critical solution temperatures (LCSTs) and cross‐linked, to determine the effect, adsorption preparation temperature has on the resultant film. The films prepared at temperatures below the LCST are smoother, thinner, and more hydrophilic than those prepared above. Human pulmonary microvascular endothelial cell (HPMEC) adhesion and proliferation are superior on the films produced below the polymers LCST compared to those produced above. Cells sheets are detached by simply lowering the ambient temperature to below the LCST. Transmission electron, scanning electron, and light microscopies indicate that the detached HPMEC sheets maintain their integrity.
Cell sheet transplantation is a key tissue engineering technology. A vascular endothelial growth factor (VEGF)‐releasing fiber mat is developed for the transplantation of multilayered cardiomyocyte sheets. Poly(vinyl alcohol) fiber mats bearing poly(lactic‐co‐glycolic acid) nanoparticles that incorporate VEGF are fabricated using electrospinning and electrospray methods. Six‐layered cardiomyocyte sheets are transplanted with a VEGF‐releasing mat into athymic rats. After two weeks, these sheets produce thicker cardiomyocyte layers compared with controls lacking a VEGF‐releasing mat, and incorporate larger‐diameter blood vessels containing erythrocytes. Thus, local VEGF release near the transplanted cardiomyocytes induces vascularization, which supplies sufficient oxygen and nutrients to prevent necrosis. In contrast, cardiomyocyte sheets without a VEGF‐releasing mat do not survive in vivo, probably undergo necrosis, and are reduced in thickness. Hence, these VEGF‐releasing mats enable the transplantation of multilayered cardiomyocyte sheets in a single procedure, and should expand the potential of cell sheet transplantation for therapeutic applications.
Poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) brushes, which are known to suppress protein adsorption and prevent cell attachment, are reported here to possess interesting and tunable thermoresponsive behavior, if the brush thickness is reduced or the grafting density is altered. PDEGMA brushes with a dry ellipsometric thickness of 5 ± 1 nm can be switched from cell adherent behavior at 37 °C to cell nonadherent at 25 °C. This behavior coincides with the temperature‐dependent irreversible adsorption of fibronectin from phosphate saline buffer and proteins present in the cell culture medium, as unveiled by surface plasmon resonance measurements. Unlike for tissue culture polystyrene reference surfaces, swelling of the PDEGMA chains below the lower critical solution temperature results in the absence of paxillin and actin containing cellular filaments responsible for cell attachment. These tunable properties of very thin homopolymer PDEGMA brushes render this system interesting as an alternative thermoresponsive layer for continuous cell culture or enzyme‐free cell culture systems.
Graphene oxide (GO) has received increasing attention in bioengineering fields due to its unique biophysical and electrical properties, along with excellent biocompatibility. The application of GO nanoparticles (GO‐NPs) to engineer self‐renewal and differentiation of human fetal neural stem cells (hfNSCs) is reported. GO‐NPs added to hfNSC culture during neurosphere formation substantially promote cell‐to‐cell and cell‐to‐matrix interactions in neurospheres. Accordingly, GO‐NP‐treated hfNSCs show enhanced self‐renewal ability and accelerated differentiation compared to untreated cells, indicating the utility of GO in developing stem cell therapies for neurogenesis.
The authors report a method to prepare cell‐laden, cell‐sized microparticles from various materials suitable for individual applications. The method includes a piezoelectric inkjetting technology and a horseradish peroxidase (HRP)‐catalyzed crosslinking reaction. The piezoelectric inkjetting technology enables production of cell‐laden, cell‐sized (20–60 μm) droplets from a polymer aqueous solution. The HRP‐catalyzed crosslinking of the polymer in the ejected solution enables production of spherical microparticles from various materials. Superior cytocompatibility of the microencapsulation method is confirmed from the viability and growth profiles of normal murine mammary gland epithelial cells.
Cell‐free approaches to in situ tissue engineering require materials that are mechanically stable and are able to control cell‐adhesive behavior upon implantation. Here, the development of mechanically stable grafts with non‐cell adhesive properties via a mix‐and‐match approach using ureido‐pyrimidinone (UPy)‐modified supramolecular polymers is reported. Cell adhesion is prevented in vitro through mixing of end‐functionalized or chain‐extended UPy‐polycaprolactone (UPy‐PCL or CE‐UPy‐PCL, respectively) with end‐functionalized UPy‐poly(ethylene glycol) (UPy‐PEG) at a ratio of 90:10. Further characterization reveals intimate mixing behavior of UPy‐PCL with UPy‐PEG, but poor mechanical properties, whereas CE‐UPy‐PCL scaffolds are mechanically stable. As a proof‐of‐concept for the use of non‐cell adhesive supramolecular materials in vivo, electrospun vascular scaffolds are applied in an aortic interposition rat model, showing reduced cell infiltration in the presence of only 10% of UPy‐PEG. Together, these results provide the first steps toward advanced supramolecular biomaterials for in situ vascular tissue engineering with control over selective cell capturing.
Multivalent aptamer–siRNA conjugates containing multiple mucin‐1 aptamers and BCL2‐specific siRNA are synthesized, and doxorubicin, an anthracycline anticancer drug, is loaded into these conjugates through intercalation with nucleic acids. These doxorubicin‐incorporated multivalent aptamer–siRNA conjugates are transfected to mucin‐1 overexpressing MCF‐7 breast cancer cells and their multidrug‐resistant cell lines. Doxorubicin‐incorporated multivalent aptamer–siRNA conjugates exert promising anticancer effects, such as activation of caspase‐3/7 and decrease of cell viability, on multidrug‐resistant cancer cells because of their high intracellular uptake efficiency. Thus, this delivery system is an efficient tool for combination oncotherapy with chemotherapeutics and nucleic acid drugs to overcome multidrug resistance.
The strand material in extrusion‐based bioprinting determines the microenvironments of the embedded cells and the initial mechanical properties of the constructs. One unmet challenge is the combination of optimal biological and mechanical properties in bioprinted constructs. Here, a novel bioprinting method that utilizes core–shell cell‐laden strands with a mechanically robust shell and an extracellular matrix‐like core has been developed. Cells encapsulated in the strands demonstrate high cell viability and tissue‐like functions during cultivation. This process of bioprinting using core–shell strands with optimal biochemical and biomechanical properties represents a new strategy for fabricating functional human tissues and organs.
Cell sorting is important for cell biology and regenerative medicine. A visible light‐responsive cell scaffold is produced using gold nanoparticles and collagen gel. Various kinds of cells are cultured on the visible light‐responsive cell scaffold, and the target cells are selectively detached by photoirradiation without any cytotoxicity. This is a new image‐guided cell sorting system.
Affinity‐based cell separation is label‐free and highly specific, but it is difficult to efficiently and gently release affinity‐captured cells due to the multivalent nature of cell‐material interactions. To address this challenge, we have developed a platform composed of a capture substrate and a cell‐releasing molecular trigger. The capture substrate is functionalized with a cell‐capture antibody and a coiled‐coil A . The cell‐releasing molecular trigger B ‐PEG (polyethylene glycol), a conjugate of a coiled‐coil B and polyethylene glycol, can drive efficient and gentle release of the captured cells, because A / B heterodimerization brings B ‐PEG to the substrate and PEG chains adopt extended conformations and break nearby multivalent antibody‐biomarker interactions. No enzymes or excessive shear stress are involved, and the released cells have neither external molecules attached nor endogenous cell‐surface molecules cleaved, which is critical for the viability, phenotype, and function of sensitive cells.
A gold standard for esophagus reconstruction is not still available. The present work aims to design a polymer patch combining synthetic polylactide‐co‐polycaprolacton and chitosan biopolymers, tailoring patch properties to esophageal tissue characteristics by a temperature‐induced precipitation method, to get multilayered patches (1L, 2L, and 3L). Characterization shows stable multilayered patches (1L and 2L) by selection of copolymer type, and their M w. In vitro investigation of the functional patch properties in simulated physiologic and pathologic conditions demonstrates that the chitosan layer (patch 3L) decreases patch stability and cell adhesion, while improves cell proliferation. Patches 2L and 3L comply with physiological esophageal pressure (3–5 kPa) and elongation (20%).
Aggregation‐caused quenching (ACQ) is a general phenomenon that is faced by traditional fluorescent polymers. Aggregation‐induced emission (AIE) is exactly opposite to ACQ. AIE molecules are almost nonemissive in their molecularly dissolved state, but they can be induced to show high fluorescence in the aggregated or solid state. Incorporation of AIE phenomenon into polymer design has yielded various polymers with AIE characteristics. In this review, the recent progress of AIE polymers for biological applications is summarized.
This article reports the behavior of embryonic neural stem cells on a hydrogel that combines cationic, non‐specific cell adhesion motifs with glycine‐arginine‐glycine‐aspartic acid‐serine‐phenylalanine (GRGDSF)‐peptides as specific cell adhesion moieties. Therefore, three hydrogels are prepared by free radical polymerization that contains either a GRGDSF‐peptide residue ( P1 ), amino ethylmethacrylate as a cationic residue ( P2 ), or a combination of both motifs ( P3 ). For each gel, cross linker concentrations of 8 mol% is used to have a comparable gel stiffness of 8–9 kPa. The cell experiments indicate a synergistic effect of the non‐specific, cationic residues, and the specific GRGDSF‐peptides on embryonic neural stem cell behavior that is especially pronounced in the cell adhesion experiments by more than doubling the number of cells after 72 h when comparing P3 with P2 and is less pronounced in the proliferation and differentiation experiments.
Strong injectable chitosan thermosensitive hydrogels can be created, without chemical modification, by combining sodium hydrogen carbonate with another weak base, namely, beta‐glycerophosphate (BGP) or phosphate buffer (PB). Here the influence of gelling agent concentration on the mechanical properties, gelation kinetics, osmolality, swelling, and compatibility for cell encapsulation, is studied in order to find the most optimal formulations and demonstrate their potential for cell therapy and tissue engineering. The new formulations present up to a 50‐fold increase of the Young's modulus after gelation compared with conventional chitosan‐BGP hydrogels, while reducing the ionic strength to the level of iso‐osmolality. Increasing PB concentration accelerates gelation but reduces the mechanical properties. Increasing BGP also has this effect, but to a lesser extent. Cells can be easily encapsulated by mixing the cell suspension within the hydrogel solution at room temperature, prior to rapid gelation at body temperature. After encapsulation, L929 mouse fibroblasts are homogeneously distributed within scaffolds and present a strongly increased viability and growth, when compared with conventional chitosan‐BGP hydrogels. Two particularly promising formulations are evaluated with human mesenchymal stem cells. Their viability and metabolic activity are maintained over 7 d in vitro.
Although chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western world, it remains incurable with conventional chemotherapeutic agents. Tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) is an antitumor candidate in cancer therapy. This study examines the proapoptotic effects of poly(propylene imine) (PPI) glycodendrimers modified with the maltotriose residues (PPI‐G4‐OS‐Mal‐III and PPI‐G4‐DS‐Mal‐III) on the TNF family in CLL cells. The combination of an understanding of the signaling pathways associated with CLL and the development of a molecular profiling is a key issue for the design of personalized approaches to therapy. Gene expression is determined with two‐color microarray 8 × 60K. The findings indicate that PPI‐G4‐OS/DS‐Mal‐III affect gene expression from the TRAIL apoptotic pathway and exert a strong effect on CLL cells comparable with fludarabine. Dendrimer‐targeted technology may well prove to bridge the gap between the ineffective treatment of today and the effective personalized therapy of the future.
Biocompatible and antibacterial hydrogels have received increasing attention for preventing local bacterial infections. In this study, a type of polysaccharide hydrogels is prepared via the Schiff‐based reaction at physiological conditions. The gelation time and mechanical property of the hydrogels are found to be dependent on the polysaccharide concentration and the polysaccharide weight ratio. 3‐(4,5‐Dimethyl‐thiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay and live/dead assay indicate that the hydrogels display nontoxicity in vitro. After subcutaneous injection into rats, the hydrogels exhibit an acceptable biocompatibility in vivo. Furthermore, the bacterial inhibition tests by shaking flask method and agar disc‐diffusion method demonstrate that the ceftriaxone‐sodium‐loaded hydrogels have remarkable antibacterial properties in vitro. The in vivo anti‐infective tests further display that the antibiotic‐loaded hydrogels display excellent anti‐infective efficacies in both superficial and deep tissue infection. Consequently, the injectable and biocompatible polysaccharide hydrogels may serve as promising platforms for localized, sustained delivery of antibiotics for preventing local infections.
The authors report on series of side‐chain smectic liquid crystal elastomer (LCE) cell scaffolds based on star block‐copolymers featuring 3‐arm, 4‐arm, and 6‐arm central nodes. A particular focus of these studies is placed on the mechanical properties of these LCEs and their impact on cell response. The introduction of diverse central nodes allows to alter and custom‐modify the mechanical properties of LCE scaffolds to values on the same order of magnitude of various tissues of interest. In addition, it is continued to vary the position of the LC pendant group. The central node and the position of cholesterol pendants in the backbone of ε‐CL blocks (alpha and gamma series) affect the mechanical properties as well as cell proliferation and particularly cell alignment. Cell directionality tests are presented demonstrating that several LCE scaffolds show cell attachment, proliferation, narrow orientational dispersion of cells, and highly anisotropic cell growth on the as‐synthesized LCE materials.
Here, it is demonstrated that X‐ray nanotomography with Zernike phase contrast can be used for 3D imaging of cells grown on electrospun polymer scaffolds. The scaffold fibers and cells are simultaneously imaged, enabling the influence of scaffold architecture on cell location and morphology to be studied. The high resolution enables subcellular details to be revealed. The X‐ray imaging conditions were optimized to reduce scan times, making it feasible to scan multiple regions of interest in relatively large samples. An image processing procedure is presented which enables scaffold characteristics and cell location to be quantified. The procedure is demonstrated by comparing the ingrowth of cells after culture for 3 and 6 days.
A simple and rapid process for multiscale printing of bioinks with dot widths ranging from hundreds of microns down to 0.5 μm is presented. The process makes use of spontaneous surface charges generated pyroelectrically that are able to draw little daughter droplets directly from the free meniscus of a mother drop through jetting (“p‐jet”), thus avoiding time‐consuming and expensive fabrication of microstructured nozzles. Multiscale can be easily achieved by modulating the parameters of the p‐jet process. Here, it is shown that the p‐jet allows us to print well‐defined adhesion islands where NIH‐3T3 fibroblasts are constrained to live into cluster configurations ranging from 20 down to single cell level. The proposed fabrication approach can be useful for high‐throughput studies on cell adhesion, cytoskeleton organization, and stem cell differentiation.
Hemocompatibility and cytocompatibility of biomaterials codetermine the success of tissue engineering applications. DNA, the natural component of our cells, is an auspicious biomaterial for the generation of designable scaffolds with tailorable characteristics. In this study, a combination of rolling circle amplification and multiprimed chain amplification is used to generate hydrogels at centimeter scale consisting solely of DNA. Using an in vitro rotation model and fresh human blood, the reaction of the hemostatic system on DNA hydrogels is analyzed. The measurements of hemolysis, platelets activation, and the activation of the complement, coagulation, and neutrophils using enzyme‐linked immunosorbent assays demonstrate excellent hemocompatibility. In addition, the cytocompatibility of the DNA hydrogels is tested by indirect contact (agar diffusion tests) and material extract experiments with L929 murine fibroblasts according to the ISO 10993‐5 specifications and no negative impact on the cell viability is detected. These results indicate the promising potential of DNA hydrogels as biomaterials for versatile applications in the field of regenerative medicine.
