Hiroyuki Tsujimoto

The anti-adhesive effect of thermally cross-linked gelatin film and its influence on the intestinal anastomosis in canine models.

To generate a more effective and safer anti-adhesive material, we have developed a new type of thermally cross-linked gelatin film. In this study, we preclinically examined the anti-adhesive efficacy of this film and evaluated the possibility applying the film safely onto fresh intestinal anastomoses, compared with hyaluronate and carboxymethyl-cellulose (HA/CMC) film. Using a canine adhesion model, the degree of adhesion for each film was evaluated by adhesion scoring systems and histological observation. Three weeks after surgery, only the gelatin film showed significantly superior anti-adhesive effects compared to the control (no treatment), in particular, exhibiting excellent re-peritonization. Next, in a canine anastomosis model, the anastomoses were wrapped directly by each film and the bursting pressures of the anastomoses were examined 3 and 7 days after surgery. The gelatin film did not significantly affect either the bursting pressures or the healing process, compared with the control. However, the HA/CMC film significantly decreased the bursting pressures measured at 3 days after surgery. In conclusion, the thermally cross-linked gelatin film had satisfactory anti-adhesive effects with excellent re-peritonization. It could be safely applied to intestinal anastomoses without decreasing the bursting pressures. The gelatin film is considered to be quite favorable as an anti-adhesive material.

Development of a novel antiadhesive material, alginate flakes, ex vivo and in vivo

PurposePostoperative intra-abdominal adhesion sometimes causes significant morbidity. The aim of this study was to compare the efficacy of our newly developed antiadhesive material, alginate flakes, to the most commonly used combination of hyaluronic acid and carboxymethyl cellulose film.MethodsSodium alginate was formed into a gel, powder, or flakes. In the ex vivo study, these different alginate forms were attached onto pig skin and their antisolubility properties in saline and attachment stability were compared. In the in vivo study, a rat surgical adhesion model was used to study the properties of the alginates, and the rats were euthanized on day 14 after surgery. The efficacy of the antiadhesive materials was evaluated using an adhesion scoring system, and the locations that were treated with the antiadhesives were histologically examined.ResultsIn the alginate groups, the alginate flakes were superior with respect to the antisolubility and the attachment stability ex vivo as well as with respect to the antiadhesive efficacy in vivo. The adhesion score was almost the same as that observed in the alginate flake and cellulose film groups.ConclusionsWe developed an alginate flake material and demonstrated its antiadhesive effects both ex vivo and in vivo. This is the first reported study using this flake-like material, which has a unique characteristic in that it can be applied by spraying in compressed air. Alginate flakes may therefore be especially useful in the field of laparoscopic surgery.

Endocrine differentiation of rat enterocytes in long-term three-dimensional co-culture with intestinal myofibroblasts.

The proliferation and differentiation of the small intestinal epithelium depends on the microenvironment surrounding the stem cells, such as intestinal subepithelial myofibroblasts. Although there have been many culture studies of intestinal epithelial–mesenchymal interaction, a culture which allows long-term observations has been difficult. This study investigated the influence of intestinal subepithelial myofibroblasts on the proliferation and differentiation of intestinal epithelial cells with a relatively long-term observation of 3xa0wk using a 3D co-culture system. Cultured rat intestinal subepithelial myofibroblasts, obtained from the duodenum, were embedded in collagen gel and cells from the rat intestinal epithelial cell line IEC-6 seeded onto it. Histologic sections of the cell-embedded gels were made and histochemical and immunohistochemical examinations were carried out in conjunction with expression analysis of the pancreatic duodenal homeobox 1 (pdx-1) transcription factor in IEC-6 cells. The IEC-6 cells showed increased proliferation and displayed characteristic endocrine features when co-cultured with rat intestinal subepithelial myofibroblasts, arranging themselves into multilayer structures and becoming cuboidal, with abundant cytoplasm and oval nuclei. Some IEC-6 cells were immunohistochemically positive for chromogranin A and glicentin. They also expressed the pdx-1 transcription factor at both the mRNA and protein levels. The number and percentage of chromogranin A-positive cells increased with culture time, whereas no increase was observed in cells cultured without rat intestinal subepithelial myofibroblasts. The present study using a long-term 3D co-culture model has obtained evidence of the participation of intestinal subepithelial myofibroblasts in enteroendocrine differentiation, supported by the expression of pdx-1 and glicentin production.

Regeneration and Functional Recovery of Intrapelvic Nerves Removed During Extensive Surgery by a New Artificial Nerve Conduit: A Breakthrough to Radical Operation for Locally Advanced and Recurrent Rectal Cancers

PurposeIn the current strategy against locally advanced and recurrent rectal cancers possibly involving intrapelvic nerves, there has been a serious dilemma between extensive surgery and limited surgery. The former can attain high tumor curability by sacrificing the nerve functions while the latter prioritizes the patient quality of life by preserving the nerve functions but with a compromised curability. Here we present a new surgical strategy for locally advanced and recurrent rectal cancers, which realize both high tumor curability and good quality of life.MethodsA new artificial nerve conduit (polyglycolic acid collagen tube) developed by in site tissue engineering technology was applied to recovery the disturbed functions after removing the nerves from 11 patients undergoing extensive surgery for intrapelvic advanced or recurrent colorectal cancers. The reconstructed nerves included eight autonomic nerves which are essential for the genitourinary function and three somatic nerves which control the sensation and mobility of the legs.ResultsOut of ten cases followed up more than 2xa0years and evaluated fully, eight including two report cases showed a functional recovery of the disturbed autonomic and somatic nerves clinically. The nerve function started to recover from 3 to 6xa0months after the operation and continued to improve with times. No specific complications associated with the nerve repair have been noted.ConclusionsThe new strategy utilizing the nerve conduit can be a breakthrough in radical operations for locally advanced and recurrent rectal cancers to resolve the problems between tumor curability and the patient quality of life.

Biological properties of a thermally crosslinked gelatin film as a novel anti-adhesive material: Relationship between the biological properties and the extent of thermal crosslinking

In order to prevent postoperative adhesion and the related complications, a thermally crosslinked gelatin (TCG) film was developed and the basic biological properties were examined, paying special attention to the relationship between these properties and the extent of crosslinking of the film. The gelatin films crosslinked thermally for five different time periods (0, 1, 3, 8, and 14 hours) were developed and the following tests were performed. Regarding the material characterization of the films, the water content, the water solubility, and the enzymatic degradation for collagenase were found to be closely related to the duration of thermal crosslinking. In an in vitro study conducted to examine the cell growth of fibroblasts cultured on the films, the degree of cell growth, except no crosslinked film, was less than that observed in the control group, thus suggesting that such effects of the films on fibroblast cell growth may be related with their anti-adhesive effects. In in vivo tests, the films crosslinked for longer time periods (3, 8, and 14 hours) were retained for longer after being implanted into the abdominal cavity in rats and showed a significant anti-adhesive effect in the rat cecum adhesion models, indicating that the biodegradability and anti-adhesive effects of the TCG films depend on the duration of thermal crosslinking. In order to develop useful and effective anti-adhesive gelatin film, it is very important to optimize duration of the thermal crosslinking.

Development of gelatin flakes, a new type of anti-adhesive material: a preliminary study of in vivo rat adhesion models

To overcome the problems associated with sheet- or film-type anti-adhesive materials, we developed a new type of anti-adhesive material, gelatin flakes. We made two types of gelatin flakes with or without thermal cross-linking, and preliminarily examined their basic properties and the anti-adhesive efficacy using a rodent adhesion model. Both types of the gelatin flakes rapidly turned into gel and tightly attached the injured surfaces, absorbing the moisture and blood, when applied onto the abraded sites of rats. In addition, these flakes could be sprayed into the desired area by compressed air through a device with a long, thin tube, which could be used in laparoscopic surgery. The anti-adhesive effects of both types of gelatin flakes were similar, and both types were significantly superior compared to the non-treated group. Although further investigations are necessary, the gelatin flakes have unique and useful properties and satisfactory anti-adhesive effects, which indicate that they may be applicable in laparoscopic surgery.

Two-layer sheet of gelatin: A new topical hemostatic agent

BACKGROUND/OBJECTIVEnUncontrolled surgical bleeding is associated with increased morbidity, mortality, and hospital cost. Topical hemostatic agents available today have problems controlling hemostatic effects; furthermore, their handling is difficult and they are unsafe.nnnMETHODSnWe devised a new hemostatic agent comprising gelatin sponge and film designed to be applied to the bleeding site, thereby creating a topical hemostatic agent made of gelatin alone. The gelatin was prepared by alkali treatment to eliminate viral activity. Hemostatic effects, surgical handling, and tissue reactions of the materials, namely a two-layer sheet of gelatin, TachoSil, and gelatin sponge, were evaluated using 21 dogs spleens.nnnRESULTSnThe two-layer gelatin sheet and gelatin sponge exhibited superior hemostatic effects (100% hemostasis completed) compared with TachoSil (0-17% hemostasis). The gelatin matrix immediately absorbed blood flowing from wounds and activated the autologous components in the absorbed blood that promoted coagulation at the bleeding site. The two-layer gelatin sheet had the best surgical handling among the evaluated materials. Materials made of gelatin were associated with fewer inflammatory reactions compared with materials of TachoSil.nnnCONCLUSIONnThe two-layer sheet of gelatin is a useful topical agent because of its superior hemostatic effects and usability, and is associated with a lower risk of transmitting diseases and inflammatory reactions.

Anti‐adhesive effects of a newly developed two‐layered gelatin sheet in dogs

Adhesion after pelvic surgery causes infertility, ectopic pregnancy, and ileus or abdominal pain. The materials currently available for clinical use are insufficient. The purpose of this study was to develop an anti‐adhesive material that overcomes the limitations of conventional anti‐adhesive agents.

Reduction of air leaks in a canine model of pulmonary resection with a new staple-line buttress

OBJECTIVEnRecently, linear staplers have been used frequently in thoracic surgery; however, air leakage from the staple line is still unresolved. Various buttress materials have been developed to prevent air leakage, but performance is still not satisfactory. We are therefore developing a new material, consisting of calcium alginate nonwoven fabric covered with sodium alginate sponge.nnnMETHODSnThirty-three beagle dogs were divided into 7 groups, and each underwent thoracotomy. Right middle lobe incision was performed with a linear stapler and 1 of the following buttress methods: group A, no buttress; group B, polyglycolic acid nonwoven fabric; group C, fibrin glue alone; group D, polyglycolic acid nonwoven fabric with fibrin glue; group E, polyglycomer sheet; group F, new alginate material; and group G, polyglycolic acid nonwoven fabric plus new alginate material. Burst pressures were measured under mechanical ventilation management.nnnRESULTSnBurst pressures were 12.0 ± 6.8 cm H(2)O in group A, 31.3 ± 6.6 cm H(2)O in group B, 13.9 ± 3.8 cm H(2)O in group C, 26.9 ± 2.8 cm H(2)O in group D, 24.8 ± 1.8 cm H(2)O in group E, 48.5 ± 4.9 cm H(2)O in group F, and 54.2 ± 12.4 cm H(2)O in group G. F and G group pressures reached the target of 40 to 50 cm H(2)O and were significantly higher than those of the 5 conventional groups (Pxa0<xa0.0005)nnnCONCLUSIONSnThis alginate buttress should be effective for preventing air leakage during operations because it has both sealant and bolster effects working in conjunction.

The influences of a novel anti-adhesion device, thermally cross-linked gelatin film on peritoneal dissemination of tumor cells: The in vitro and in vivo experiments using murine carcinomatous peritonitis models: INFLUENCES OF GELATIN FILM ON PERITONEAL DISSEMINATION

To create anti-adhesive materials to be more effective and safer, we developed a thermally cross-linked gelatin film that showed superior anti-adhesive effects with excellent peritoneal regeneration. However, it may act as a convenient scaffold for tumor cell growth, thereby accelerating peritoneal dissemination when used in surgery for abdominal tumors. In this study, we tried to clarify this issue using mouse carcinomatous peritonitis models. First, we examined the in vitro tumor cell growth of mouse B16 melanoma or Colon26 cells on the gelatin film or the conventional hyarulonate/carboxymethylcellulose film. Tumor cell growth on each film was significantly lower than that of the control (no film). Next, we conducted the following in vivo experiments: After the parietal peritoneum was partially removed and covered with each film or without any film, mice were inoculated intraperitoneally with B16 melanoma or Colon26/Nluc cells expressing NanoLuc luciferase gene. At 7 days after the operation, we measured the weight of B16 melanoma tumors or the NanoLuc activity of Colon26/Nluc cells using in vivo imaging at the injured sites. There were no significant differences in the weight of the tumors and the NanoLuc activity among the three groups. We also observed the survival time of mice receiving the same operation and treatments. There was no significant difference in the survival time among the three groups. These results suggest that the gelatin film will likely not accelerate peritoneal dissemination as a convenient scaffold for tumor cell growth when used in surgery for abdominal tumors.