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

AIM: 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. METHODS: The adhesion prevention effects of three methods - a two-layered sheet composed of gelatin film and gelatin sponge, Seprafilm and INTERCEED - were evaluated in 37 dogs. Anti-adhesive effects were investigated macroscopically and microscopically in a cauterized uterus adhesion model. Cell growth on the materials in vitro using human peritoneal mesothelial cells, fibroblasts and uterine smooth muscle cells were also evaluated. RESULTS: The two-layered gelatin sheet had significantly superior anti-adhesive effects compared to the conventional materials (Seprafilm and INTERCEED). A single-cell layer of mature mesothelium formed three weeks after surgery in the gelatin group. Peritoneum regeneration in the Seprafilm and INTERCEED groups was delayed and incomplete in the early phase. Little inflammation around the materials occurred and cell growth was significantly proliferated with the gelatin sheet. CONCLUSION: The anti-adhesive effects of a two-layered gelatin sheet were superior to conventional agents in a cauterized canine uterus model, demonstrating early regeneration of the peritoneum, little inflammation and material endurance. The newly developed two-layered gelatin sheet is a useful option as an anti-adhesive agent for deeply injured and hemorrhagic sites.

Tissue engineering of small intestinal tissue using collagen sponge scaffolds seeded with smooth muscle cells.

In a previously reported attempt to regenerate small intestine with autologous tissues, collagen scaffolds were used without cell seeding or with autologous mesenchymal stem cell seeding. However the regenerated intestine lacked a smooth muscle layer. To accomplish regeneration of a smooth muscle layer, this present study used collagen scaffolds seeded with the smooth muscle cells (SMC) in a canine model. Autologous SMC were isolated from stomach wall and cultured. Two types of scaffolds were fabricated: in SMC (+), cultured SMCs were mixed with collagen solution and poured into a collagen sponge; and in SMC (-), SMCs were omitted. Both scaffolds were implanted into defects of isolated ileum as a patch graft. Animals were euthanized at 4, 8, and 12 weeks; for the last time point, the ileal loop had been reanastomosed at 8 weeks. At 12 weeks, the SMC (-) group showed a luminal surface covered by a regenerated epithelial cell layer with very short villi; however only a thin smooth muscle layer was observed, representing the muscularis mucosae. In the SMC (+) group, the luminal surface was covered completely by a relatively well-developed epithelial layer with numerous villi. Implanted SMCs were seen in the lamina propria and formed a smooth muscle layer. Thus, we concluded that collagen sponge scaffolds seeded with autologous SMCs have a potential for small intestine regeneration.

The tissue-engineered vascular graft using bone marrow without culture.

OBJECTIVE: To overcome the shortcomings of current vascular grafts, tissue-engineering methods have been applied to cardiovascular regions. We previously reported the creation of a tissue-engineered vascular graft by using vascular mixed cells. However, the cost and manpower for harvesting and culturing the cells was too burdensome. To overcome these drawbacks, we have developed a new method for creating a tissue-engineered vascular graft by using bone marrow cells, which can be obtained easily and used immediately, without cell culture. METHODS: Biodegradable polymers seeded with different types of cells (group V, cultured venous cells; group B, bone marrow cells without culture; and group C, non-cell-seeded graft [as control]) were implanted into the inferior venae cavae of dogs. The grafts were explanted at 4 weeks and assessed histologically and biochemically. RESULTS: In the histologic examination, a regular layer of Masson-staining collagen fiber and a layer of factor VII-stained endothelial and ant-alpha-smooth muscle cell antigen-immunoreactive cells stained in groups V and B like native vascular tissue, whereas no such stained regular lining was detected in group C. A 4-hydroxyproline assay in group C showed significantly lower levels than in groups V and B or native tissue ( P < .05). The DNA content of the tissue-engineered vascular graft tended to be higher in group C than in groups V and B or in native tissue. CONCLUSIONS: In the creation of tissue-engineered vascular grafts, the method of using bone marrow cells seems to be useful and superior to that of using vascular cells because bone marrow cells can be used directly, without culture.

Endocrine cell and nerve regeneration in autologous in situ tissue-engineered small intestine.

BACKGROUND: The purpose of this study was to regenerate a larger size of small intestinal tissue than that of our previous study and to evaluate the regeneration of the endocrine cells (ECC) and nerve system of autologous tissue-engineered small intestine. The effect of implantation of large numbers of smooth muscle cells (SMC) for the regeneration of small intestine was also investigated. METHODS: Two types of scaffolds with different cell densities were fabricated: low density (LD) of SMC in the scaffold and high density (HD) of SMC in the scaffold. Both scaffolds were implanted into defects of isolated ileum in a canine model. Animals were sacrificed at 8, 12, 18, and 24 weeks. RESULTS: The area of engineered small intestine in the HD group was four times larger than that in the LD group, although that was smaller in size than the original size of the defect. There were no significant changes in the thickness of regenerated smooth muscle layer (SML) in the LD and HD groups. The numbers of endocrine cells gradually increased after implantation. At 18 weeks of regeneration, the number of ECC reached levels comparable to that of normal mucosa. The nerve fibers extended to the center of the graft area and were observed in regenerated SML and regenerated villi at 24 weeks. CONCLUSIONS: The ECC and nerve fibers were regenerated in autologous in situ tissue-engineered small intestine. Seeding a large number of SMC was not sufficient for the regeneration of the small intestine in a tubular configuration.