Randomized clinical trial of an elastomeric sealant for hemostasis in thoracic aortic surgery.

OBJECTIVES: This study aimed to demonstrate the efficacy and safety of a newly developed elastomeric sealant, which does not require any blood coagulation system to exert its effect, during thoracic aortic surgery. METHODS: This is a multicenter, randomized study conducted in six hospitals in Japan. A total of 81 patients undergoing replacement surgery of a thoracic aortic aneurysm using cardiopulmonary bypass were randomized with a ratio of 2-:1 for those patients designated to receive the sealant (Group S, 54 patients) or those without the usage of the sealant (Group C, 27 patients). The primary endpoints were bleeding from each anastomosis at two time points: (1) immediately before applying protamine and (2) 15 min after applying protamine. The patients were followed for 6 months. RESULTS: The number of anastomoses checked for bleeding was 196 in Group S and 117 in Group C. Before protamine sulfate administration, complete hemostasis was obtained in 155 anastomoses (79%) in Group S compared to 45 anastomoses (38%) in Group C (p < 0.001). Fifteen minutes after the administration of protamine sulfate infusion, bleeding stopped completely in 173 anastomoses (88%) in Group S and in 71 anastomoses (61%, p < 0.001) in Group C. Between the two groups, there were no marked differences in the patient background or in the incidence of major adverse events. CONCLUSIONS: The sealant is effective in achieving hemostasis, even under fully heparinized conditions. The novel sealant is safe and effective in thoracic aortic surgery, one of the most demanding surgical situations for hemostasis.

Vascular endothelial growth factor-bound stents: application of in situ capture technology of circulating endothelial progenitor cells in porcine coronary model.

OBJECTIVES: We evaluated the in vivo performance of a newly devised vascular endothelial growth factor (VEGF)-bound stent in a porcine coronary model. BACKGROUND: An anti-CD34 antibody-bound stent, which captures endothelial progenitor cells (EPCs) to accelerate tissue formation, did not reduce intimal hyperplasia. By targeting the VEGF receptor, which is expressed on endothelial-lineage cells, we developed VEGF-bound stents that may enable selective capture of EPCs followed by rapid endothelialization. METHODS: Metallic stents were first coated with poly-(ethylene-co-vinyl alcohol), and then chemically bound with either VEGF or anti-CD34 antibody. These stents were placed in porcine coronary arteries for up to 14 days. Stent surface was evaluated by immunohistochemistry and by scanning electron microscope (SEM). RESULTS: After 2-day stenting with VEGF-bound stents, small populations of KDR (VEGF receptor-2)-positive cells adhered to the stent struts. After 7- and 14-day stenting, struts were fully covered with newly regenerated tissue. SEM images showed that the uniform tissue formed on struts was morphologically similar to native endothelium and was continuously connected with adjacent native endothelium. On the other hand, for the anti-CD34 antibody-bound stents, stent struts were rapidly covered by newly generated tissue that consisted of multicellular aggregates. CONCLUSIONS: Compared with anti-CD34 antibody-bound stents, VEGF-bound stents provide highly selective capture of EPCs, followed by rapid formation of intact endothelium tissue at an early period of stenting. These results suggest that VEGF-bound stents could represent a promising therapeutic option for cardiovascular stenting, although further long-term follow-up experiment with double-blinded fashion is needed prior to clinical application.

[Development of hemostatic sealant for arterial anastomosis; clinical application].

For the purpose of examining the clinical applicability of a newly developed surgical sealant, animal experiments were performed, and clinical trial was followed. In animal experiments, several animal models, including carotid artery anastomosis model and coronary artery bypass grafting model were undertaken. In each model, complete hemostasis of the anastomoses using four simple interrupted sutures, was obtained. In addition, elastomeric property of the sealant prevented thinning of the arterial wall. The clinical trial performed in patients with thoracic aortic surgery showed significantly better hemostasis even under heparinized condition. Based on these excellent results, clinical usage of the sealant was approved.

[Development of elastameric sealant designed for arterial field].

The development of a reliable surgical sealant specific for arterial tissues has been long awaited. In this article, first the "ideal" adhesion mechanism formulated from biomechanical concept is proposed for ensured hemostasis in dissected arteries with pulsatile flow. An urethane prepolymer prepared along the design criteria is viscous liquid. Due to its high water absorbility and high reactivity with water, the sealant applied to vascular tissues becomes an elastomer within several minutes. When the sealant was applied to dissected canine abdominal arteries with 3 stay sutures, followed by declamping 5 minutes, neither bleeding nor detrimental effect on tissue morphogenesis was observed. This sealant is being ready to the market.

Surface design for in situ capture of endothelial progenitor cells: VEGF-bound surface architecture and behaviors of cultured mononuclear cells.

In situ capture of endothelial progenitor cells (EPCs) in the arterial bloodstream may allow the creation of a functioning endothelium on the luminal surfaces of implanted cardiovascular devices. Our strategy is to use highly biospecific interaction between the cell-surface marker and surface-bound protein. The target-cell marker defined is vascular endothelial growth factor (VEGF) receptor, which is exclusively expressed on endothelial lineage cells. The candidate surface-bound proteins are VEGF and anti-VEGF receptor (VEGF-R1 and VEGF-R2) antibodies, which were covalent-bound on poly(ethylene-co-vinyl alcohol) bearing a high-surface density of hydroxyl groups. Incubating human mononuclear cells on these substrates affected the histochemical expression of cell-surface markers specific for EPCs and endothelial cells (ECs). The VEGF-bound surface significantly increased the number of cells expressing both VEGF receptors after 1 or 2 weeks of culture, whereas both anti-VEGF receptor antibody-bound substrates did not affect the expression of the surface markers, and cells on these surfaces were eventually died. These results indicate that, among the three candidate molecules, VEGF is best able to capture EPCs and induces their differentiation. Additionally, a pilot study of surface architecture of stents and small-diameter artificial grafts was conducted for an ongoing implantation study in a porcine model.

Arterial shear stress augments the differentiation of endothelial progenitor cells adhered to VEGF-bound surfaces.

Our ongoing studies show that vascular endothelial cell growth factor (VEGF)-bound surfaces selectively capture endothelial progenitor cells (EPCs) in vitro and in vivo, and that surface-bound VEGF stimulates intracellular signal transduction pathways over prolonged culture periods, resulting in inductive differentiation of EPCs. In this article, we investigated whether simulated arterial shear stress augments the differentiation of EPCs adhered to a VEGF-bound surface. Human peripheral blood-derived mononuclear cells adhered to a VEGF-bound surface were exposed to 1 day of shear stress (15 dynes/cm(2), corresponding to shear load in arteries). Shear stress suppressed the expression of mRNAs encoding CD34 and CD133, which are markers for EPCs, and augmented the expression of mRNAs encoding CD31 and von Willebrand factor (vWF) as well as vWF protein, which are markers for endothelial cells (ECs). Shear stress enhanced expression of ephrinB2 mRNA, a marker for arterial ECs, but did not significantly change expression of EphB4 mRNA, a marker for venous ECs. Focused protein array analysis showed that mechanotransduction by shear stress activated the p38 and MAPK pathways in EPCs. Thus, arterial shear stress, in concert with surface-bound VEGF, augments the differentiation of EPCs. These results strongly support previous observation of rapid differentiation of EPCs captured on VEGF-bound stents in a porcine model.

Hydrodynamic shear-stress-dependent retention of endothelial and endothelial progenitor cells adhered to vascular endothelial growth factor-fixed surfaces.

The luminal surfaces of small-diameter artificial vascular grafts must be fully endothelialized to be nonthrombogenic following implantation. To achieve this goal, we have attempted to capture circulating endothelial progenitor cells (EPCs) in situ on the luminal surfaces of implanted grafts. We examined potential receptor-ligand pairs that promote selective and tight adhesion of EPCs using a radial flow chamber comprising three regions, each containing a specific protein-bound substrate: fibronectin (FN) for integrin, and vascular endothelial growth factor (VEGF) and anti-Flk-1 antibody for VEGF receptor. In the presence of shear stress, the greatest retention of endothelial cells and EPCs was observed with FN followed by VEGF and then anti-Flk-1 antibody. Regardless of the bound protein, cell adhesion increased with larger areas of cell adhesion and enhanced cell spreading; the latter was also greatest with FN followed by VEGF and then anti-Flk-1 antibody. The distribution of vinculin-a key protein in focal adhesion plaques-in adherent endothelial cells was examined using total internal reflection fluorescence microscopy; FN-bound surfaces resulted in larger areas of adhesion and more focal adhesion plaques compared with surfaces bound with VEGF. On the other hand, examining these parameters relative to the area of cell adhesion revealed that VEGF-bound surfaces resulted in larger focal adhesion areas and greater fluorescence signals, both of which indicate increased resistance to shear stress. We also discuss in situ capturing of EPCs on surfaces bound with VEGF or anti-Flk-1 antibody, with the goal of creating endothelialized small-diameter vascular grafts.

Luminal surface design of electrospun small-diameter graft aiming at in situ capture of endothelial progenitor cell.

If endothelial progenitor cells (EPCs), which circulate in blood flow, are captured on the luminal surface of an implanted artificial graft and sooner or later proliferate to form a fully endothelialized surface, such a small-diameter artificial graft must exhibit a high patency rate. This study aimed at designing a luminal surface of elastomeric electrospun mesh graft, which is capable of selective capture of EPCs under arterial flow and has a high antithrombogenic potential until full endothelization is achieved. The designed luminal surface layer is composed of a photopolymerized gelatin gel layer that enables the release of impregnated heparin and selective adhesion of circulating EPCs via complexation between surface-fixed vascular endothelial growth factor (VEGF) and cellular VEGF receptor. Human mononuclear cells seeded and cultured on such a gel layer expressed endothelial cell surface markers. Confocal laser scanning microscopy observation revealed that VEGF is highly surface-enriched, and heparin is homogeneously distributed in the gel layer. A continuously slow release of heparin was observed. Thus, a prototype luminal surface was fabricated on electrospun segmented polyurethane tubes for in vivo study.

Articular cartilage tissue engineering based on a mechano-active scaffold made of poly(L-lactide-co-epsilon-caprolactone): In vivo performance in adult rabbits.

Our previous studies showed that a mechano-active scaffold made of poly(L-lactide-co-epsilon-caprolactone) (PLCL) exhibited a high potential to realize the formation of a functional, engineered cartilage in vitro. This animal study therefore was designed to investigate the feasibility of repairing on osteochondral defect with the use of bone marrow-derived mesenchymal stem cells (BMSCs) incorporated with a PLCL scaffold. Rabbit BMSCs, isolated and subsequently cultured in monolayer, were seeded into a porous PLCL scaffold sponge following an implantation onto a full-thickness osteochondral defect (diameter of 4.5 mm, depth of 5 mm) that was artificially created on the medial femoral condyles at a high load-bearing site on a rabbit's knee joint. Time-dependent healing of the defect was evaluated by macroscopic, histological examinations at both 3- and 6-month-implantations, respectively. A PLCL sponge incorporated with BMSCs exhibited sufficient structural support, resulting in new osteochondral tissue regeneration: a physiologically well-integrated subchondral bone formation, a hyaline cartilage-like morphology containing chondrocytes surrounded by abundant cartilaginous matrices. In addition, quantitative biochemical assays also demonstrated high potential for the synthesis of sulfated glycosaminoglycan and collagen, both of which are biomolecules essential to extracelluar matrix in normal cartilage tissue. In contrast, defects filled with cell-free PLCL scaffold or left empty showed a very limited potential for regeneration. Our findings suggest that a composite of PLCL-based sponge scaffold and BMSCs promote the repair of osteochondral defects at high load-bearing sites in adult rabbits.

Experimental use of an elastomeric surgical sealant for arterial hemostasis and its long-term tissue response.

OBJECTIVES: Reliable suture line hemostasis should improve the outcome of aortic surgery. We examined the hemostatic effect and the tissue response of a novel elastomeric surgical sealant. METHODS: Using porcine internal carotid arteries, we performed 16 end-to-end anastomoses with four stitches of simple interrupted sutures under full heparinization. The anastomoses were divided into two groups (eight anastomoses per group). Either novel sealant or fibrin glue was applied. The amount of bleeding was measured during the 30 s period after removing the vascular clamp. In a separate experiment, we applied the novel sealant around the abdominal aorta of rabbits (n=6) to assess the effect of the elastomeric property of the sealant on arterial wall histology. For comparison, we applied cyanoacrylate, which has no elastomeric property (n=6). A histological study was performed three months after the operation. RESULTS: The novel sealant prevented arterial bleeding. The amount of bleeding from the anastomoses applied with novel sealant and fibrin glue was 0.12+/-0.03 g vs. 91.8+/-16.5 g, respectively (P<0.001). Thinning of the rabbit aortic wall was observed in the cyanoacrylate-treated abdominal aorta, whereas no thinning was observed in the novel sealant group. Histological examination revealed neither cell death nor necrosis in the novel sealant group. CONCLUSIONS: The novel sealant effectively prevented arterial bleeding from the anastomosis under full heparinization. In addition, the elastomeric property of the sealant prevented thinning of the aortic wall. The novel sealant may be a promising hemostatic agent for arterial anastomosis.

Nanoscale elongating control of the self-assembled protein filament with the cysteine-introduced building blocks.

Self-assembly of artificially designed proteins is extremely desirable for nanomaterials. Here we show a novel strategy for the creation of self-assembling proteins, named "Nanolego." Nanolego consists of "structural elements" of a structurally stable symmetrical homo-oligomeric protein and "binding elements," which are multiple heterointeraction proteins with relatively weak affinity. We have established two key technologies for Nanolego, a stabilization method and a method for terminating the self-assembly process. The stabilization method is mediated by disulfide bonds between Cysteine-residues incorporated into the binding elements, and the termination method uses "capping Nanolegos," in which some of the binding elements in the Nanolego are absent for the self-assembled ends. With these technologies, we successfully constructed timing-controlled and size-regulated filament-shape complexes via Nanolego self-assembly. The Nanolego concept and these technologies should pave the way for regulated nanoarchitecture using designed proteins.

Lymphocyte adhesion and interactions with biomaterial adherent macrophages and foreign body giant cells.

To characterize the effects of adherent macrophages and biomaterial surface chemistries on lymphocyte adhesion and activation, lymphocytes were co-cultured with monocytes alone and together, directly and separated by a porous membrane transwell on hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/cationic biomaterial surfaces. Surface adherent cells were quantitatively analyzed after 3 days utilizing immunofluorescence and phase contrast imaging. After periods of 3, 7, and 10 days, secreted interferon-gamma (IFN-gamma) was quantified by ELISA. Limited direct biomaterial-adherent lymphocytes were identified regardless of the presence of macrophages or foreign body giant cells (FBGC). The majority of adherent lymphocytes, which were T cells (>95%) rather than natural killer cells, predominantly interacted with adherent macrophages and FBGCs; greater than 90% were interacting on surfaces with higher levels of adherent macrophages and FBGCs and greater than 55% were interacting on surfaces with lower levels of macrophages and FBGCs. The hydrophilic/anionic surface promoted higher levels of macrophage- and FBGC-adherent lymphocytes but was nonselective for lymphocyte subtype interactions. The hydrophilic/neutral surface was selective for CD4+ T lymphocyte interactions while the hydrophobic surface was selective for CD8+ T lymphocyte interactions. IFN-gamma was produced in direct and indirect co-cultures but not in lymphocyte- and monocyte-only cultures suggesting that lymphocytes are activated via macrophage-derived cytokines rather than direct biomaterial contact. Direct lymphocyte interactions with adherent macrophages/FBGCs enhanced IFN-gamma production relative to indirect co-cultures. These results suggest that lymphocytes prefer interactions with adherent macrophages and FBGCs, resulting in lymphocyte activation, and these interactions can be influenced by biomaterial surface chemistries.

Instability of self-assembled monolayers as a model material system for macrophage/FBGC cellular behavior.

Novel self-assembled monolayers (SAMs) designed to present homogenous surface chemistries were utilized to further investigate the material surface chemistry dependent macrophage and foreign-body giant cell (FBGC) behaviors, including macrophage adhesion, fusion, and apoptosis. Contact angle analysis revealed instabilities in the --CH(3) and --COOH terminated SAM surfaces upon incubation in serum-free media (SFM) at 37 degrees C or under dry, room temperature conditions. Further analysis indicated that the --CH(3) terminated SAM surface degraded rapidly within 2 h and loss of sufficient SAM units to be comparable to the gold (Au) control surface, within 24 h of incubation in SFM at 37 degrees C. After 5 days of incubation in SFM at 37 degrees C, the contact angles for the --COOH terminated SAM surfaces increased markedly. AFM analysis confirmed the desorption of --CH(3) terminated SAM molecules from the surface with increased roughness and marked appearance of peaks and valleys within 2 h. A decrease in the thickness of the --COOH terminated SAM surface also suggests molecular desorption over time. No significant changes in contact angle or AFM analyses were observed on the --OH terminated SAM surfaces. Cellular adhesion decreased more rapidly on the Au control and --CH(3) terminated SAM surfaces in comparison to the other surfaces. However by day 10, cellular adhesion, fusion, and apoptosis were comparable on all SAM surfaces and the Au control. These studies suggest that SAM surfaces may not be suitable for long-term studies where material dependent properties are investigated.

Lymphocyte/macrophage interactions: biomaterial surface-dependent cytokine, chemokine, and matrix protein production.

The role of lymphocytes in the biological response to synthetic polymers is poorly understood despite the transient appearance of lymphocytes at the biomaterial implant site. To investigate cytokines, chemokines, and extracellular matrix (ECM) proteins produced by lymphocytes and macrophages in response to biomaterial surfaces, human peripheral blood monocytes and lymphocytes were co-cultured on polyethylene terephthalate (PET)-based material surfaces displaying distinct hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/cationic chemistries. Antibody array screening showed the majority of detected proteins are inflammatory mediators that guide the early inflammatory phases of wound healing. Proteomic ELISA quantification and adherent cell analysis were performed after 3, 7, and 10 days of culture. IL-2 and IFN-gamma were not detected in any co-cultures suggesting lack of lymphocyte activation. The hydrophilic/neutral surfaces increased IL-8 relative to the hydrophobic PET surface (p < 0.05). The hydrophilic/anionic surfaces promoted increased TNF-alpha over hydrophobic and cationic surfaces and increased MIP-1beta compared to hydrophobic surfaces (p < 0.05). Since enhanced macrophage fusion was observed on hydrophilic/anionic surfaces, the production of these cytokines likely plays an important role in the fusion process. The hydrophilic/cationic surface promoted IL-10 production and increased matrix metalloproteinase (MMP)-9/tissue inhibitor of MMP (TIMP) relative to hydrophilic/neutral and anionic surfaces (p < 0.05). These results suggest hydrophilic/neutral and anionic surfaces promote pro-inflammatory responses and reduced degradation of the ECM, whereas the hydrophilic/cationic surfaces induce an anti-inflammatory response and greater MMP-9/TIMP with an enhanced potential for ECM breakdown. The study also underscores the usefulness of protein arrays in assessing the role of soluble mediators in the inflammatory response to biomaterials.

Matrix metalloproteinases and their inhibitors in the foreign body reaction on biomaterials.

Matrix metalloproteinases (MMPs) can degrade structural components within the extracellular matrix and at the cellular surface producing changes in cellular behavior (i.e., adhesion and migration) and subsequent pathological responses (i.e., the foreign body reaction and wound healing). We continue to study the foreign body reaction that occurs following biomaterial implantation by investigating secretory responses of biomaterial-adherent macrophages and foreign body giant cells (FBGCs) as directed by material surface chemistry and further this research by determining whether secreted MMPs play a role in macrophage adhesion and fusion. We have identified numerous MMPs and their tissue inhibitors (TIMPs) in in vitro cell-culture supernatants using antibody arrays and quantified select MMP/TIMPs with ELISAs. MMP-9 concentrations were significantly greater than both TIMP-1 and TIMP-2 on all materials. The ratios of MMP-9/TIMP-1 and MMP-9/TIMP-2 increased with time because of an increase in MMP-9 concentrations over time, while the TIMP concentrations remained constant. Total MMP-9 concentrations in the supernatants were comparable on all materials at each timepoint, while TIMP-1 and TIMP-2 concentrations tended to be greater on hydrophilic/anionic surfaces. Analysis of the MMP/TIMP quantities produced per cell revealed that the hydrophilic/neutral surfaces, which inhibited macrophage adhesion, activated the adherent macrophages/FBGCs to produce a greater quantity of MMP-9, TIMP-1, and TIMP-2 per cell. Pharmacological inhibition of MMP-1,-8,-13, and -18 reduced macrophage fusion without affecting adhesion, while inhibitors of MMP-2,-3,-9, and -12 did not affect adhesion or fusion. These findings demonstrate that material surface chemistry does modulate macrophage/FBGC-derived MMP/TIMP secretion and implicates MMP involvement in macrophage fusion.

Elastomeric surgical sealant for hemostasis of cardiovascular anastomosis under full heparinization.

PURPOSE: We developed a novel surgical sealant, a viscous diisocyanated prepolymer, applicable to arterial hemostasis. The purpose of this study is to evaluate hemostatic effect of this surgical sealant under heparinized conditions. METHODS: The effectiveness of this sealant was verified by applying it to the end-to-end anastomosis of canine carotid arteries. Five mongrel dogs were used. After a complete heparinization, the carotid arteries were clamped, divided, and end-to-end anastomoses were performed with four simple interrupted sutures. The sealant was coated on the anastomosis. After 5 min the clamps were removed and the hemostatic effect was evaluated. Three dogs were immediately subjected to macroscopic evaluation. Two dogs were subjected to angiography after 3 months and 16 months, respectively. RESULTS: No bleeding occurred in any of the anastomoses immediately after the removal of the clamp. Macroscopic finding revealed no leakage of the sealant into the lumen. Carotid angiography revealed patent anastomoses without stenosis. CONCLUSION: A novel surgical sealant exhibited rapid and potent hemostatic effect on a moisturized tissue under full heparinization.

Proteomic analysis and quantification of cytokines and chemokines from biomaterial surface-adherent macrophages and foreign body giant cells.

Implantation of biomaterial devices results in the well-known foreign body reaction consisting of monocytes, macrophages, and foreign body giant cells (FBGCs) at the material/tissue interface. We continue to address the hypothesis that material surface chemistry modulates the phenotypic expression of these cells. Utilizing our human monocyte culture system, we have used surface-modified polymers displaying hydrophobic, hydrophilic, and/or ionic chemistries to determine the cytokines/chemokines released from biomaterial-adherent macrophages/FBGCs. This study broadens our approach by using proteomic analysis to identify important factors expressed by these cells and further quantifies these molecules with ELISAs. Proteomic profiles changed over time suggesting that the adherent macrophages underwent a phenotypic switch. Macrophage/FBGC-derived proinflammatory cytokines, IL-1beta and IL-6, decreased with time, while the anti-inflammatory cytokine, IL-10, gradually increased with time. Resolution of the inflammatory response was also demonstrated by a decrease in chemoattractant IL-8 and MIP-1beta production with time. Material-dependent macrophage/FBGC activation was analyzed using cytokine/chemokine production and cellular adhesion. Monocyte/macrophage adhesion was similar on all surfaces, except for the hydrophilic/neutral surfaces that showed a significant decrease in cellular density and minimal FBGC formation. Normalizing the ELISA data based on the adherent cell population provided cytokine/chemokine concentrations produced per cell. This analysis showed that although there were fewer cells on the hydrophilic/neutral surface, these adherent cells were further activated to produce significantly greater amounts of each cytokine/chemokine tested than the other surfaces. This study clearly presents evidence that material surface chemistry can differentially affect monocyte/macrophage/FBGC adhesion and cytokine/chemokine profiles derived from activated macrophages/FBGCs adherent to biomaterial surfaces.

Dynamic force spectroscopy of the specific interaction between the PDZ domain and its recognition peptides.

To characterize the molecular basis of specific interactions of PDZ proteins, dynamic force spectroscopy (DFS) for the PDZ protein Tax-interacting protein-1 (TIP-1) and its recognition peptide (PDZ-pep) derived from beta-catenin was performed using an atomic force microscope (AFM), together with measurement of thermodynamic and kinetic parameters using surface plasmon resonance (SPR). The unbinding force of this pair was measured under different conditions of AFM tip-retraction velocity. The relationship between the unbinding force and the logarithmic force-loading rate, that is, the dynamic force spectrum, exhibited two different rate regimes, for each of which the forces increased linearly with the force-loading rate. On the basis of the theoretical treatment of the Bell-Evans model, the positions of two different activation barriers in the reaction coordinate and dissociation rate constants in each barrier were evaluated from slopes and x-intercepts of the two linear regimes (first barrier: 0.04 nm and 1.10 x 10 s(-1); second barrier: 0.21 nm and 2.77 x 10(-2) s(-1), respectively). Although two-step unbinding kinetics between TIP-1 and PDZ-pep was suggested from the DFS analysis, SPR results showed single-step dissociation kinetics with a rate constant of 2.89 x 10(-1) s(-1). Different shapes of the free energy profile of the unbinding process were deduced from each result of DFS and SPR. The reason for such topographic differences in the energy landscape is discussed in relation to the differences in the pathways of forced unbinding and spontaneous dissociation.

Photo-iniferter-based thermoresponsive block copolymers composed of poly(ethylene glycol) and poly(N-isopropylacrylamide) and chondrocyte immobilization.

A series of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM)-poly(ethylene glycol) (PEG) block copolymers with various PNIPAM contents and copolymer architectures, such as linear, four-armed and eight-armed configurations, were prepared by iniferter-based photopolymerization of dithiocarbamylated PEGs (DC-PEGs) under ultraviolet (UV)-light irradiation. The increase in monomer/DC-PEG feed ratio resulted in an increase in both the molecular weight and PNIPAM content of copolymers. The measurement of the optical transmittances of aqueous solutions of PNIPAM-PEG block copolymers determined the lower critical solution temperatures (LCSTs) of block copolymers, which ranged from 31.3 to 34.0 degrees C. LCST decreased with increasing block length of PNIPAM and with the formation of a branched architecture. Rabbit chondrocytes were immobilized and cultured in a three-dimensional (3D) gel composed of PNIPAM-PEG block copolymer at 37 degrees C. Gels prepared from copolymers with higher PNIPAM contents at higher concentrations appeared to exhibit a minimal decrease in both cell number and cell viability during a 7-day culture. Cell viability dependencies on material and formulation parameters and the potential use of PNIPAM-PEG block copolymers as an in situ formable scaffold for an engineered cartilage tissue were discussed.

Force measurement for antigen-antibody interaction by atomic force microscopy using a photograft-polymer spacer.

To determine the intermolecular force on protein-protein interaction (PPI) by atomic force microscopy (AFM), a photograft-polymer spacer for protein molecules on both surfaces of the substrate and AFM probe tip was developed, and its effectiveness was assessed in a PPI model of a pair of human serum albumin (HSA) and its monoclonal antibody (anti-HSA). A carboxylated photoiniferter, N-(dithiocarboxy)sarcosine, was derivatized on both surfaces of the glass substrate and AFM probe tip, and subsequently water-soluble nonionic vinyl monomers, N,N-dimethylacrylamide (DMAAm), were graft-polymerized on them upon ultraviolet light irradiation. DMAAm-photograft-polymerized spacers with carboxyl groups at the growing chain end but with different chain lengths on both surfaces were prepared. The proteins were covalently bound to the carboxyl terminus of the photograft-polymer chain using a water-soluble condensation agent. The effects of the graft-spacer length on the profile of the force-distance curves and on the unbinding characteristics (unbinding force and unbinding distance) were examined in comparison with those in the case of the commercially available poly(ethylene glycol) (PEG) spacer. The frequency of the nonspecific adhesion force profile was markedly decreased with the use of the photograft spacers. Among the force curves detected, a high frequency of single-peak curves indicating the unbinding process of a single pair of proteins and a very low frequency of multiple-peak profiles were observed for the photograft spacers, regardless of the graft chain length, whereas a high frequency of no-force peaks was noted. These observations were in marked contrast with those for the PEG spacer. The force peak values determined ranged from 88 to 94 pN, irrespective of the type of spacer, while the standard deviation of force distribution observed for the photograft spacer was lower than that for the PEG spacer, indicating that the photograft spacers provide a higher accuracy of force determination.