Synthetic polymer-tissue adhesion using an ultrasonic scalpel.

BACKGROUND: Interface phenomena such as encapsulation and formation of dead space around implanted biomaterials lower biomaterial performance. To advance tissue adhesive technology, understanding the interactions between tissue (collagen) and polymer is indispensable. Adhesion between tissue and polymer was studied using an ultrasonically activated scalpel (UAS). METHODS: The Harmonic Scalpel was used as an ultrasonically activated scalpel for polymer and tissue adhesion. A piece of porcine aorta and a polymeric film were layered and placed between the blades of the Harmonic Scalpel. Then the samples were gripped with 20 kgf of force for 1-10 s to adhere the porcine aorta and polymeric films. The adhesion was characterized by macro- and microscopic observation, thermographic analysis, and measurement of bonding strength, static contact angle (SCA), and surface properties. RESULTS: Cellulose, vinylon, polyethylene terephthalate, nylon, and Pellethane could be bonded to the aorta. Bonding was not observed for the polyethylene, polypropylene, polyvinyl chloride, or polytetrafluoroethylene. This suggests that the existence of functional groups such as hydroxyl, carbonyl, carboxyl, and amide groups in the polymer structure are key factors in adhesion. Harmonic Scalpel modification of the polyethylene surface during corona discharge treatment further indicated that the functional groups of the polymers are one of the essential factors for tissue adhesion. The temperatures of adhesion were 90-150 °C for the polymers, and the melting temperatures (Tm) were 193-306 °C. This suggests that adhesion was formed by the interaction between the melted polymer surface and the tissue collagen. CONCLUSION: Both polar functional groups and adequate thermal characteristics are necessary for polymers to bond with tissues. These findings should be useful for the development of novel polymers that can be bonded to living tissues with UAS treatment, which can be applied for endoscopic surgery.

Changes in the morphology of cell-size liposomes in the presence of cholesterol: formation of neuron-like tubes and liposome networks.

Spontaneous changes in the morphology of cell-size liposomes (dioleoylphosphatidylcholine, DOPC and egg PC) as model cells were investigated in the presence of cholesterol. Tube structures and liposome networks connected by the tubes were observed in the presence of 5-30% cholesterol by dark-field and laser-scanning microscopy. Furthermore, in the presence of more than 40 mol% of cholesterol, the tubes disappeared and changed to small liposomes. Thus, cholesterol induced a morphological change in giant liposomes from tubes to small liposomes. These phenomena may be related to the role of cholesterol in the morphological changes in living cells such as neurons.

Formation of embryoid bodies by mouse embryonic stem cells on plastic surfaces.

Mouse embryonic stem (ES) cells were cultured on artificial polymeric biomembranes with a phospholipid polymer (phosphatidylcholine, PC) surface. ES cells aggregated to form an embryoid body (EB) on the PC surface immediately after seeding. Single EBs formed on the PC surface after 3 d, and their size was depended on the initial number of cells that were seeded. In contrast, many small EBs with a nonuniform shape formed on a conventional hydrophobic nontreated polystyrene surface. RT-PCR assays of the EBs indicated that cell-cell interactions were enhanced in EBs that formed on the PC surface compared with EBs that formed on the polystyrene surface. The transcription factor Pax6, which is a marker of the differentiation of ES cells to neurons, was not expressed in EBs that formed on the PC surface; however, EBs that formed on the polystyrene surface did express Pax6, indicating that they were undergoing differentiation into neurons. When stimulated with retinoic acid (an inducer of differentiation into neurons), EBs on the PC surface expressed Pax6. We also observed that the adhesion of ES cells to the PC surface was reduced. Thus, the formation of large EBs on the PC surface was due to enhanced cell-cell interaction and inhibition of nonspecific differentiation to neurons.

A nonthrombogenic gas-permeable membrane composed of a phospholipid polymer skin film adhered to a polyethylene porous membrane.

Polymer membranes are widely used in biomedical applications such as hemodialysis, membrane oxygenator, etc. When the membranes come in contact with blood or body fluids, protein adsorption and cell adhesion occur rapidly. Nonspecific protein adsorption and cell adhesion on the membranes induce not only various bio-rejections but also a decrease in their performance. We hypothesized that a blood compatible gas-permeable membrane could be prepared from polyethylene (PE) porous membranes modified with phospholipid polymers. In this study, poly[(2-methacryloyloxyethyl phosphorylcholine) (MPC)-co-dodecyl methacrylate] (PMD) skin film adhered to a PE porous membrane (PMD/PE porous membrane) was prepared. Elution of PMD was not detected meaning that the PMD film did not detach from the PE porous membrane even after soaking in water for more than 6 months. The permeation coefficient of oxygen gas through the PE membrane with the adhered PMD containing more than 0.20 mole fraction of the MPC unit, was the same as that of the original PE porous membrane. The PMD surface effectively reduced biofouling. We concluded that the PMD/PE porous membrane is useful as a novel membrane oxygenator due to its excellent gas-permeability and blood compatibility.

Conformation of poly(sodium ethacrylate) in solution studied by small-angle X-ray scattering.

The pH-induced conformational transition of poly(sodium ethacrylate) PNaEA in aqueous solution, which occurs between a compact form at low charge-density and an extended coil at high charge-density, was studied by small-angle X-ray scattering and the structure at an each conformational state was analyzed and compared with the corresponding one of poly(sodium methacrylate) PNaMA. The conformational transition for PNaEA induced a remarkable change in the scattering data plotted in the form of the Kratky plot. By comparing the scattering data with theoretical scattering functions, it was clarified that the structures of the compact form and the extended coil are well mimicked by a swollen gel having a network structure and by a wormlike chain, respectively. Although such a structure of the extended coil of PNaEA is similar to the corresponding one of PNaMA, the structure of the compact form of PNaEA is different from the corresponding one of PNaMA, which is still represented by a wormlike chain in a Theta medium.

Control of cell function on a phospholipid polymer having phenylboronic acid moiety.

We synthesized a water-insoluble phospholipid polymer bearing a phenylboronic acid moiety (PMBV), which induces cell adhesion through a specific interaction with the glycoprotein, fibronectin. Surface plasmon resonance analysis revealed that fibronectin was adsorbed on the PMBV surface. When fibroblasts were cultured on the PMBV surface, the cells adhered and proliferated normally while showing a spherical morphology. In addition, the adherent cells were able to detach after the addition of sugar molecules, which bound to phenylboronic acid through an exchange reaction. The cell cycle of adherent cells was evaluated with the embedded HeLa-Fucci cells by using a fluorescent ubiquitination-based cell cycle indicator. The cell-cycle analysis by fluorescence microscopy indicated that the adherent HeLa-Fucci cells tended to converge to the G1 phase. The differentiation of mesenchymal stem cells to chondrocytes was accelerated on PMBV in the presence of bone morphogenetic protein-2. We concluded that PMBV is a useful surface in experiments for assessing cellular function and differentiation.

Selective biorecognition and preservation of cell function on carbohydrate-immobilized phosphorylcholine polymers.

To obtain synthetic materials capable of selectively recognizing proteins and cells, and preserving their functions, biomembrane mimetic polymers having a phospholipid polar group and carbohydrate side chains were designed. Poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-2-lactobionamidoethyl methacrylate (LAMA)] (PMBL) was synthesized and coated on substrates by solvent evaporation. Selective binding of galactose-recognized lectin, RCA120, to a PMBL surface was investigated by measurement of surface plasmon resonance. The binding of RCA120 to the PMBL surface was confirmed by a remarkable change in resonance angle. The apparent affinity constant of RCA120 to PMBL3.0 (3.0 mol % LAMA unit in the feed) per LAMA unit was 2.77 x 10(5) M(-1). When a glucose-recognized lectin, concanavalin A, was in contact with PMBL, no change in the resonance angle was observed, and any nonspecific fouling of protein on PMBL was effectively reduced. Cells of the human hepatocellular liver carcinoma cell line (HepG2) having asialoglycoprotein receptors (ASGPRs) were seeded on polymer surfaces. On poly(BMA) (PBMA), many adherent cells were observed and were well-spread with monolayer adhesion, but cell adhesion was reduced on poly(MPC-co-BMA) (PMB). HepG2 adhesion was observed on PMBL because the cell has ASGPRs; the number of cells adhering to the PMBL polymer surfaces increased with an increase in the density of galactose residues on the surface. In contrast, adhesion of NIH-3T3 cells to PMBL was reduced in a manner similar to that on PMB because the NIH-3T3 cells did not have ASGPRs. Cell adhesion to the PMBL surface was well-regulated by ligand-receptor interactions. Furthermore, some of the cells adhering to the PMBL surface had a spheroid form, and similarly shaped spheroids were scattered on the surface. Although poly(BMA-co-LAMA) (PBL) has galactose residues, the adherent cells were spread in a manner similar to those on PBMA. The MPC units in PMBL contribute to make a spheroid formation of HepG2 cells. The amount of albumin secreted from a cell was compared with the chemical structure of the substrate. The spheroid shaped cells cultured on the PMBL surface secreted much more albumin than did the spreading cells that adhered to the PBMA. In conclusion, the biomembrane mimetic carbohydrate-immobilized phosphorylcholine polymers produced a suitable surface for biorecognition and preservation of cell function.

A stereocomplex platform efficiently detecting antigen-antibody interactions.

Ultrathin poly(methyl methacrylate) (PMMA) stereocomplex films with macromolecularly double-stranded regular nanostructures were prepared by layer-by-layer assembly of isotactic and syndiotactic PMMAs on solid surfaces. Antibodies were immobilized through the Fc region-capturing protein A, which had been physically adsorbed on the complex film, and the binding of antigens to immobilized antibodies was quantitatively investigated by the quartz crystal microbalance technique. Greater amounts of protein A with native forms were adsorbed on the complex film than those on conventional single-component PMMA films. Antibodies with high target-binding activities were also immobilized on the complex film. A greater amount of antigens could be detected on the complex film. The activity of protein A was maintained on the complex for a long time even within a dried state. The mechanism for the preservation of protein native forms on the complex surface was speculated by analyzing the physical adsorption of proteins with various secondary structures. Stereocomplex films can be utilized as novel coating nanomaterials for efficiently detecting protein-protein interactions.

Selective cell attachment to a biomimetic polymer surface through the recognition of cell-surface tags.

Reactive phosphorylcholine polymers, which can recognize biosynthetic cell-surface tags, were synthesized to control cell attachment. Human promyelocytic leukemia cells (HL-60) with unnatural carbohydrates as cell-surface tags were harvested by treatment with N-levulinoylmannosamine (ManLev). The attachment of ManLev-treated HL-60 cells to 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers with hydrazide groups was studied. HL-60 cells, which are nonadhesive, did not attach to any polymer surface without ManLev treatment. In contrast, ManLev-treated HL-60 cells attached to a poly[MPC-co-n-butyl methacrylate (BMA)-co-methacryloyl hydrazide (MH)] (PMBH) surface following 15 min of incubation. The cells that attached to the PMBH surface retained their native morphology and viability for 24 h of incubation. On the other hand, approximately half of the HL-60 cells that attached to the poly(BMA-co-MH) (PBH) surface died. These results suggest that MH units in the polymer act as anchors for cell attachment and MPC units help to preserve cell viability on a polymer surface. The coculture of ManLev-treated HL-60 and fluorescence-stained human uterine cervical cancer cells (HeLa) was carried out on polymer surfaces. ManLev-treated HL-60 cells specifically attached to the PMBH surface. In contrast, both HL-60 and HeLa cells were observed on the PBH surface. The control of cellular interactions with synthetic polymers may be useful for the future development of cell-integrated biosensors and biomedical devices.

Importance of a biofouling-resistant phospholipid polymer to create a heparinized blood-compatible surface.

Heparinization is believed to be one of the methods to suppress thrombus formation on blood-contacting surfaces. However, this study hypothesizes that heparinization alone might not be sufficient to provide a blood-compatible surface; that is, a surface property that resists biofouling is necessary to obtain an effective heparin-modified surface. 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers with 2-aminoethyl methacrylate (AEMA) were synthesized to immobilize heparin through ionic bonding. The primary amino groups of AEMA were considered to be the polymer surface because the zeta-potential of the surface was positive when the mole fraction of the AEMA units was above 0.2. The antithrombogenic character of the polymer surface modified with heparin was evaluated by both Lee-White and microsphere column methods. The coagulation period of human whole blood in the absence of anticoagulant in glass tubing coated with the MPC polymer was longer than that in the original glass tube. Cell adhesion was completely inhibited on the MPC polymer surface after contact with human whole blood without anticoagulant. However, many adherent blood cells were observed on poly(2-ethylhexyl methacrylate-co-AEMA) (no MPC unit) even after heparinization. These results strongly indicate that the MPC polymer is a useful substrate where the heparin works well and that the heparin-immobilized MPC polymer has superior blood compatibility to the simple MPC polymer.