Yasushi Tamada

Simple method for platelet counting

A method is proposed for counting the number of adhered platelets based on the determination of lactate dehydrogenase activity in bulk after lysis of adhered platelets. This method was compared with the widely used radioisotope labelling technique. It was concluded that the present lactate dehydrogenase method is effective in counting the adhered platelets, as no significant difference was found between the readings of two methods when commercial polymers and glass were used as samples.

Cell adhesion to plasma-treated polymer surfaces

Abstract Polymer surfaces were modified by glow discharge to study the effect of surface treatment on cell adhesion using polyethylene, polytetrafluoroethylene, poly(ethylene terephthalate), polystyrene, and polypropylene films. The surface wettability of all the films, evaluated by the water contact angle, decreased with respect to the length of plasma treatment. For each of the polymers, a different dependence of cell adhesion on the length of plasma treatment was observed, but, in each case, the optimal water contact angle for cell adhesion was approximately 70°. The X-ray photoelectron spectroscopy (XPS) of the plasma-treated surfaces using a derivatization method suggested that hydroxyl groups were primarily introduced onto the surfaces of the polymer by plasma treatment. Formation of carboxyl groups by plasma treatment was also observed from XPS, although streaming potential measurements could not identify the newly generated groups.

Cell behaviour on polymer surfaces grafted with non-ionic and ionic monomers

Following exposure to corona discharge, a polyethylene film was graft polymerized with different water-soluble monomers such as acrylamide (non-ionic), acrylic acid (anionic), 2-acrylamide-2-methyl propane sulphonic acid (anionic), styrene sulphonic acid sodium salt (anionic) and N,N-dimethylaminopropyl acrylamide (cationic). Attachment and proliferation of HeLa S3 cells were studied for grafted surfaces with different zeta potentials and contact angles. The polyethylene surface graft polymerized with styrene sulphonic acid sodium salt exhibited high cell attachment and protein adsorption, whereas the cells did not adhere to the 2-acrylamide-2-methyl propane sulphonic acid graft-polymerized surface, although both surfaces had high negative zeta potentials. Graft polymerization of acrylamide reduced the zeta potential of surface close to zero and rejected the cell attachment. The polyethylene surface became highly cell-adhesive through graft polymerization of the cationic N,N-dimethylaminopropyl acrylamide monomer, but too much grafting killed the attaching cells. Once the cells attached to a surface without being killed, they could proliferate at the same growth rate, whatever their surface zeta potential.

Experimental study of a newly developed bilayer artificial skin

A bilayer artificial skin composed of an outer layer of silicone polymer and an inner sponge layer of collagen containing chondroitin 6-sulphate was developed by modifying the technique proposed by Yannas et al. The artificial skin was placed on the skin defects on the backs of rats. Histological observation indicated that fibroblasts and capillaries infiltrated into the pores and filled in lattice spaces, resulting in synthesis of the connective tissue matrix and absorption of the original network of collagen and chondroitin 6-sulphate. Epidermal cells migrated from the edge of the wound between the two layers. Post-operative contracture in the wound with the artificial skin was significantly less than in the control.

Preparation and characterization of apatite deposited on silk fabric using an alternate soaking process

Apatite-deposited silk fabric composite materials were developed using a new alternate soaking process. The characteristics of deposited apatite were studied using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectrophotometry (FTIR), and X-ray photoelectron spectroscopy (XPS). Apatite weight increased with alternating soaking in a calcium solution [200 mM aqueous calcium chloride solution buffered with tris(hydroxymethyl) aminomethane and HCl (pH 7.4)] and a phosphate solution (120 mM aqueous disodium hydrogenphosphate) changed every hour. SEM showed that apatite deposited after 21 or more repeated soakings was over 20 microm thick. XRD showed that with alternate soakings, the apatite crystals deposited on silk fabric elongated along the c axis. FTIR and XPS indicated the existence of carbonate, HPO(4)(2-), and Na(+) ions in addition to constituent ions of hydroxyapatite. A loss of HPO(4)(2-) and Na(+) ions in the deposit upon further soaking might be associated with an increasing apatite crystallinity. Apatite deposited on silk by the alternate soaking process was a deficient apatite containing carbonate, HPO(4)(2-), and Na(+) ions as in a natural bone tissue. Thus, this apatite-silk composite material might be potentially bioactive.

Preparation of gel film from Bombyx mori silk sericin and its characterization as a wound dressing.

Sericin is a highly hydrophilic protein family acting as the glue in Bombyx mori silk. In order to apply sericin as a wound dressing, a novel sericin film named gel film was prepared by a simple process without using any chemical modifications: sericin solution was gelled with ethanol into a sheet shape and then dried. Infrared analysis revealed that the sericin gel film contained water-stable β-sheet networks formed in the gelation step. This structural feature rendered the gel film morphologically stable against swelling and gave it good handling properties in the wet state. The sericin gel film rapidly absorbed water, equilibrating at a water content of about 80%, and exhibited elastic deformation up to a strain of about 25% in the wet state. A culture of mouse fibroblasts on the gel film indicated that it had low cell adhesion properties and no cytotoxicity. These characteristics of sericin gel film suggest its potential as a wound dressing.

Sulfation of silk fibroin by chlorosulfonic acid and the anticoagulant activity

Silk fibroin (Bombyx mori) was sulfated using chlorosulfonic acid in pyridine. FT-IR spectra showed introduction of sulfate group by this reaction; NMR spectra indicated that sulfation occurred mainly at tyrosine and serine residues. Molecular size decreased and dispersed with sulfation. The molecular weight was estimated in around 20,000 by GPC using protein standards. Amino acid composition suggested that sulfated fibroin came from H-chain of fibroin; the crystal region of fibroin molecule remained in sulfated fibroin. The amount of sulfate groups increased with overall reaction time. The maximum amount was estimated in 1.0 mmol/g by acidimetric titration. Sulfation efficiency was calculated as 66.7%. Blood coagulation was prevented by 0.5 mg of sulfated fibroin in 1 ml of blood, while original fibroin did not show any effect. Anticoagulant activity of sulfated fibroin strongly depends on the amount of sulfate groups introduced. These results indicate that sulfate group introduction results in addition of anticoagulant function to silk fibroin. Sulfated fibroin is a new type of anticoagulant material having a protein backbone.

A New Method for the Modification of Fibroin Heavy Chain Protein in the Transgenic Silkworm

We constructed a new plasmid vector for the production of a modified silk fibroin heavy chain protein (H-chain) in the transgenic silkworm. The plasmid (pHC-null) contained the promoter and the 3′ region of a gene encoding the H-chain and the coding regions for the N-terminal domain and the C-terminal domain of the H-chain. For the model protein, we cloned a foreign gene that encoded EGFP between the N-terminal domain and the C-terminal domain in pHC-null and generated transgenic silkworms that produced a modified H-chain, HC-EGFP. Transgenic silkworms produced HC-EGFP in the posterior part of silk gland cells, secreted it into the lumen of the gland, and produced a cocoon with HC-EGFP as part of the fibroin proteins. N-terminal sequencing of HC-EGFP localized the signal sequence cleavage site to between positions A(21) and N(22). These results indicate that our new plasmid successfully produced the modified H-chain in a transgenic silkworm.

Sulfation of silk sericin and anticoagulant activity of sulfated sericin

—Silk sericin extracted from a cocoon was sulfated by chlorosulfonic acid. Sulfation of sericin was confirmed by FT-IR and the reaction efficiency was calculated as 44.9%. 1H-NMR suggested that sulfation mainly occurred at serine residues in the sericin molecule. The sulfated sericin was separated in three fractions by gel-filtration chromatography using Sephacryl S-200. The sulfate group content and amino-acid composition of each fraction were almost identical, while the anticoagulant activity differed for each fraction of sulfated sericin. Higher anticoagulant activity was observed for the higher-molecular-mass fraction. The anticoagulant activity of sulfated sericin was estimated at 1/10 to 1/20 of heparin.

Anticoagulant mechanism of sulfonated polyisoprenes

The influence of sulfonated polyisoprene (SPIP) on coagulation factors and human blood cells was investigated to elucidate and compare its anticoagulant mechanism with that of heparin. While the number of red cells was unaffected, the number of platelets decreased dramatically in the presence of SPIP due to aggregation. Using a synthetic peptide substrate to assay thrombin activity in the presence of its natural inhibitor, antithrombin (AT), we observed no stimulation by SPIP of AT-mediated inhibition. Nevertheless, thrombin cleavage of its natural substrate fibrinogen to fibrin peptide A was slightly inhibited. SPIP altered the electrophoretic mobility of fibrinogen and completely inhibited fibrinogen from clotting. We detected no significant influence of SPIP on factors II, VII, IX, and X, while factor XI and factors V and VIII were only slightly affected. Therefore, the main mechanism of SPIPs anticoagulant activity appears to be a strong interaction with fibrinogen and fibrin monomer, first, to prevent proteolytic conversion of the former to the latter and second, to inhibit polymerization of the fibrin monomer, once formed.