Naoki Nakajima

Photo-Induced Cytotoxicity of Water-Soluble Fullerene

Abstract The reaction of C60 with poly(ethylene glycol) having a terminal primary amino group or a mixture of ethylene diamine and poly(ethylene glycol) having terminal carboxyl groups resulted in formation of water-soluble C60 conjugates. These conjugates showed strong cytotoxicity to L929 cells upon visible light irradiation as a result of superoxide production.

Surface modification of poly(ethylene-co-vinyl alcohol) (EVA). Part I. Introduction of carboxyl groups and immobilization of collagen.

To enhance the surface biocompatibility of poly(ethylene-co-vinyl alcohol) (EVA) and high-density polyethylene (HDPE), carboxyl groups were introduced by ozone exposure. Type I collagen was immobilized onto the surface through polyion complexing. The carboxyl groups on the EVA were characterized by electron spectroscopy for chemical analysis and neutralization. The amounts of the carboxylic group and collagen increased with increases in time and temperature of exposure. Water-soluble fragments were produced by ozone exposure to EVA, and they acted as collagen crosslinkers. The differences in charge distribution of carboxyl groups affected the amount of collagen immobilization. Graft polymerization of acrylic acid was also carried out onto EVA and HDPE surfaces. The amount of collagen immobilized by graft polymerization was much higher than that by ozone exposure despite the introduction of almost the same amounts of carboxylic groups. It was suggested that the negative charge distribution influences the amount of collagen immobilized onto films.

Sutureless amniotic membrane transplantation for ocular surface reconstruction with a chemically defined bioadhesive

The purpose of this study was to evaluate the efficiency and safety of a sutureless amniotic membrane transplantation (AMT) for ocular surface reconstruction with a chemically defined bioadhesive (CDB). The CDB was synthesized from aldehyded polysaccharides and epsilon-poly(L-lysine), two kinds of medical and food additives, as starting materials. Biocompatibility assay indicated that the CDB showed excellent biocompatibility with in vitro and in vivo ocular surface tissues and most of the CDB was histologically degraded within 4 weeks. Sutureless AMT using the CDB was safely and successfully performed onto a rabbit scleral surface. Transplanted amniotic membrane (AM) evaluated by histological, electron microscopic- and immunohistochemical examination indicated that the CDB did not affect normal differentiation of the cells or the integrity of the surrounding tissue. Thus, this newly developed CDB was found to be very useful for sutureless AMT for ocular surface reconstruction, without considering the risk of infection. It has the ability to fix AM to the ocular surface for a long time-period without additional inflammation, scarring, or damage to the surrounding tissues.

Self-Degradable Bioadhesive

To improve the conventional and commercially-available medical adhesives such as cyanoacrylate, aldehyde-based, and fibrin glue, new bioadhesive has been prepared using medical and food additives as starting materials. Aldehyde groups could be easily introduced in dextran in the presence of sodium periodate in aqueous media, and the extent of the introduction could also be controlled. In vitro degradation speed of the hydrogel prepared by mixing of aldehyded dextran with ε-poly(L-lysine) at 37oC significantly varied by acetic anhydride concentration added to ε-poly(L-lysine) from < 5h to > 5 weeks. Bonding strength of the glue was 4 times higher than that of commercial fibrin glue and almost no cytotoxicity was observed, suggesting the development of novel self-degradable bioadhesive.

Biodegradable polymer coating promotes the epithelization of tissue-engineered airway prostheses

OBJECTIVE We have developed a prosthesis that includes a collagen layer for tracheobronchial reconstruction and applied it in a canine model. In previous studies luminal epithelization remained partial or rather slow because of the early disintegration of the collagen layer. We have improved this type of prosthesis by coating the luminal surface with a biodegradable polymer, which serves to protect the collagen layer. The effect of the polymer coating on the epithelization of the luminal surface of the prosthesis was examined. METHODS The main frame consisted of a polypropylene mesh tube, measuring 15 mm in inner diameter and 30 mm in length, with reinforcing rings. Collagen extracted from porcine skin was conjugated to this frame. The luminal surface was coated with a polymer, poly (L-lactic-acid-co-epsilon-caprolactone). In 5 beagle dogs the left main bronchus was replaced with this prosthesis, periodic bronchoscopic observations were conducted, and microscopic evaluations were performed. RESULTS All dogs survived until they were killed, except for 1 animal in which pneumonia developed, and this animal died at 13 months after replacement. None of the dogs showed adverse complications caused by the prosthesis. Bronchoscopic observations revealed that the polymer remained on the luminal surface for 2 weeks. The luminal surface in 4 dogs was completely covered with ciliated columnar epithelium or nonciliated squamous epithelium, and 90% epithelization was achieved in 1 dog. CONCLUSIONS The biodegradable polymer coating protected the collagen layer and promoted better epithelization. This improved epithelization on the luminal surface could therefore potentially increase the success rates in airway replacement with artificial prostheses.

Development of a New Tissue-Engineered Sheet for Reconstruction of the Stomach

We have developed tissue-engineered digestive tracts composed of collagen scaffold and an inner silicon sheet and successfully used it to repair defects in parts of the esophagus, stomach, and small intestine. However, some improvements were demanded for clinical usage because the silicon sheet presented technical difficulties for suturing and endoscopic removal. New tissue-engineered sheet (New-sheet) was composed of a single-piece and reinforced collagen scaffold with biodegradable copolymer. One beagle dog was used to evaluate whether New-sheet could withstand suturing in comparison with native digestive tracts using a tensile tester. Seven beagle dogs had a 5-cm circular defect created in the stomach. New-sheet soaked with autologous peripheral blood or bone marrow aspirate was sutured to the gastric wall. Endoscopic, histological, and immunohistochemical assessment was performed to evaluate regeneration of the stomach up to 16 weeks. Tensile strength testing showed that the mucosal side of New-sheet had strength almost equivalent to the mucosa of the esophagus (P = 0.61). Endoscopically, regeneration of the mucosa started from the circumference after 4 weeks, but a small linear ulcer was still evident at 16 weeks. The regenerated stomach shrank by 60-80% of its original size and histologically showed villous mucosa and underlying dense connective tissue. Immunohistochemically, the regenerated area expressed alpha-smooth-muscle actin but was negative for basic calponin, irrespective of the source of soaked blood. New-sheet shows sufficient strength for suturing, no dehiscence, and better biocompatibility for clinical use, although further examination will be necessary to create a functional digestive tract.

Low cytotoxic tissue adhesive based on oxidized dextran and epsilon-poly-l-lysine

A novel adhesive hydrogel consisting of dextran and epsilon-poly(L-lysine) (dextran-PL) with multiple biomedical applications was developed. Periodate oxidation in aqueous media almost stoichiometrically introduces aldehyde groups in dextran molecules, and aldehyde dextran can react with the primary amino groups in epsilon-PL (ɛ-PL) at neutral pH to form a hydrogel. The gelation time of the hydrogel can be easily controlled by the extent of oxidation in dextran and of the acylation in ɛ-PL by anhydrides. The shear adhesion strength of dextran-PL was 10 times higher than that of fibrin glue, when wet collagen sheets were selected as test specimens. The cytotoxicity of aldehyde dextran and ɛ-PL were 1000 times lower than that of glutaraldehyde and poly(allylamine). The considerably low cytotoxicity of aldehyde dextran could be ascribed to its low reactivity with amine species when compared with glutaraldehyde. In contrast, a high reactivity of amino groups in ɛ-PL was observed when compared with glycine, L-lysine, and gelatin, which could be explained by their poor dissociation at neutral pH, thus leading to low cytotoxicity.

Morphologic study and syntheses of type I collagen and fibronectin of human periodontal ligament cells cultured on poly(ethylene-co-vinyl alcohol) (EVA) with collagen immobilization.

The purpose of this study was to regenerate the human periodontal ligament (PDL) around dental implants by using a hybrid structure of cells with materials, such as PDL cells + collagen + poly(ethylene-co-vinyl alcohol) (EVA) on titanium implant. Human PDL cells were cultured on the EVA surface coated with type I collagen and the cell adhesion and extension were investigated. Furthermore, collagen type I and fibronectin syntheses were analyzed. The serum free culture was also tried, to investigate the role of collagen in detail. The results showed that: 1. Satisfactory adhesion, extension, and proliferation of the PDL cells on the EVA films coated with collagen were observed, but were not good without collagen. 2. Immunostaining of cultured PDL cells revealed the syntheses of type I collagen, when cultured on the EVA coated with collagen or conventional culture dish, though fibronectin synthesis was observed even in the EVA without collagen. 3. Only PDL cells on the EVA coated with collagen proliferated well in the absence of serum. These results indicate that our novel implant material (EVA coated with collagen) provides a possibility of PDL regeneration on dental implants.

Fusogenic activity of various water-soluble polymers

The fusogenic activity of seven water-soluble polymers was investigated using L929 cells in a monolayer state. Among these polymers, only two, poly(ethylene glycol) (PEG) and EPAN 680 were capable of inducing the membrane fusion of L929 cells. EPAN 680 is an ABA type block copolymer composed of 80% ethylene oxide(A) and 20% propylene oxide(B) sequences with a total molecular weight of 8800. Evaluation of the polymer hydrophobicity indicated that there was no clear correlation between it and the fusogenic activity of the polymer, although highly hydrophobic polymers caused cell shrinkage without membrane fusion. Differential scanning calorimetry on these polymers strongly suggested that hydration of the polymers in culture medium had a large effect on their fusogenic activity. It was concluded that the assembly of cell membranes, stabilized by water molecules, was disturbed by strong interaction with the polymer molecules having a strong hydration power, resulting in membrane fusion.

Novel Powdered Anti-adhesion Material: Preventing Postoperative Intra-abdominal Adhesions in a Rat Model

Background: Although laparoscopic surgery has decreased postoperative adhesions, complications induced by adhesions are still of great concern. The aim of this study was to investigate the anti-adhesive effects of a novel powdered anti-adhesion material that can be applied during laparoscopic surgery in comparison with other anti-adhesion materials. Methods:Our novel powdered anti-adhesion material is composed of aldehyde dextran and ε-poly(L-lysine). In 40 male rats, a 2.5×2.0-cm abdominal wall resection and cecum abrasion were performed. The rats were randomized into four groups based on the anti-adhesion treatments: normal saline; Seprafilm®; Interceed®; and novel powdered anti-adhesion material. The animals were euthanized on days 7 and 28 to evaluate the adhesion severity, area of adhesion formation, gross appearance, and pathological changes. Results: The adhesion severities on both days 7 and 28 were significantly lower for all anti-adhesion material groups compared with the normal saline group (p<0.05). Pathologically, all groups showed inflammatory cell infiltration on day 7 and complete regeneration of the peritoneum on day 28. Conclusions:Our novel powdered anti-adhesion material was found to be effective for reducing postoperative intra-abdominal adhesions and showed equivalent efficacy to commercial anti-adhesion materials.