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We studied capillary endothelial injury, as demonstrated by fibrin deposition and changes in thrombomodulin (Tm) expression, in rats receiving continuous or bolus iv lipopolysaccharide (LPS). Rats were continuously infused with iv LPS (0.1, 0.2, 0.5, or 1.0 mg/kg/hr) for up to 6 hours. Others were given a bolus iv dose of LPS (20 mg/kg), and then the same dose of saline as a continuous infusion was administered for up to 3 hours. Harvested lungs, livers, and kidneys were examined immunohistochemically for thrombomodulin expression and fibrin deposition. Tm expression began to diminish dose- and time-dependently in lung, liver, and renal peritubular capillaries within 2 to 4 hours of the start of continuous LPS administration (1.0 mg/kg/hr) and had completely disappeared by 3 hours, although Tm remained in the glomerulus. The amount of fibrin deposition observed varied with the organ, dose, and duration of treatment in rats that received continuous LPS administration, but little was deposited in the lung. After bolus LPS administration, Tm in the endothelia of lung, liver, and peritubular capillaries diminished 20 to 40 minutes after treatment and then recovered 120 to 180 minutes after treatment, but the Tm activity of the glomerulus did not change. Fibrin deposition in the capillaries was observed in the liver, glomerulus, and peritubular capillaries, but not in the lung. Endothelial injury by LPS administration is dependent on the dose of LPS and the duration of treatment. The amount of fibrin deposition differs among organs and with the duration of contact between the endothelium and the endotoxin.

The rate of biodegradation of new types of polyglycolic acid (PGA)-collagen composite tubes for nerve regeneration was evaluated in the peritoneal cavity. PGA mesh tubes with a diameter of 2 or 4 mm were coated with collagen solution and dried at room temperature. The tubes were then subjected to dehydrothermal treatment (composite tube). A 2 mm PGA-collagen composite tube filled with collagen sponge was also investigated in this study (sponge tube). Tubes with a length of 15 mm were fixed at the parietal peritoneum of BALB/c mice and excised 2 weeks and 1, 2, and 3 months after the operation. The inner areas of the excised tubes were measured microscopically. Statistical analysis was performed by one way ANOVA and Fisher's PLSD test. Although the inner areas of the 2 and 4 mm composite tubes were not maintained 1 month after the operation (62 +/- 6.8% and 21 +/- 3.8%, respectively), they were well maintained in the sponge tubes (83 +/- 6.4%). The inner areas of the sponge tubes were significantly larger than those of the composite tubes until 2 months after surgery. These results suggest that sponge tubes are more suitable than composite tubes for nerve regeneration in the peritoneal cavity.

Collagen sponges of various biodegradabilities were prepared by dehydrothermal crosslinking at 140 degrees C for different time periods. When the collagen sponges were radioiodinated and implanted subcutaneously into the back of mice, the radioactivity remaining at the implanted site decreased with time; the longer the time of dehydrothermal crosslinking, the slower the radioactivity decrement. The radioactivity following the subcutaneous implantation of collagen sponges incorporating (125)I-labeled transforming growth factor (TGF)-beta1 also decreased with time. The time profile of both the radioactivity remainings was in good accordance to each other, irrespective of the crosslinking time. This indicates that the TGF-beta1 incorporated in the sponges was released as a result of sponge biodegradation. Potential of collagen sponges incorporating 0.1 micro g of TGF-beta1 in repairing the defect of rabbit skulls was evaluated in a stress-unloaded state. Bone repairing was induced by application of the collagen sponges incorporating 0.1 micro g of TGF-beta1 whereas that of free TGF-beta1 at the same dose and TGF-beta1-free, empty collagen sponges were ineffective. The bone defect was histologically closed by the bone tissue newly formed 6 weeks after application. Bone mineral density (BMD) analysis revealed that the collagen sponge incorporating TGF-beta1 enhanced the BMD value at the bone defect to a significantly great extent compared with other agents. A maximum enhancement of BMD was observed for the collagen sponge incorporating TGF-beta1 which was prepared by dehydrothermal crosslinking for 6 h. It was concluded that the TGF-beta1 incorporated in the collagen sponge was released in a biologically active form as a result of sponge biodegradation, resulting in enhanced bone repairing at the skull defect. It is possible that for too slowly degraded sponges, the remaining physically impairs the bone repairing at the skull defect. Induction of bone repairing would not be achieved through a rapid release of TGF-beta1 from too fast-degraded sponge.

We prepared a complex membrane consisting of human amnion-derived collagen membrane and polyglycolic acid (PGA), and then gelatin was crosslinked using heat on one side of the complex membrane to prepare a membrane that can prevent adhesion (gelatin-processed PGA-human collagen membrane). Applying this membrane to a rabbit cecum-abdominal-wall-adhesion model, the prevention of adhesion and tissue regeneration were investigated. The animals were sacrificed 2 and 12 weeks after surgery and then examined. The adhesion scores in the short-term observation group (2 weeks after surgery) and long-term observation group (12 weeks after surgery) were 1.0 +/- 2.4 and 0.8 +/- 2.0, respectively, showing a significant prevention of adhesion compared to the control value of 6.3 +/- 2.5 (p < 0.01). Histologically, gelatin was not absorbed, and outgrowth of connective tissue accompanied by capillary blood vessels was observed between the sample and the cecum in the short-term observation group. In the long-term observation group, the sample was completely absorbed, and serous membrane was regenerated on the surface of connective tissue. Based on these findings, it is possible to use gelatin-processed PGA-human collagen membrane as a filling material with both an adhesion-preventing effect and tissue-regenerating function., (Copyright 2002 Wiley Periodicals, Inc.)

We investigated whether saber sheath-type tracheomalacia could be treated by the slow release of bone morphogenetic protein (BMP)-2 from a gelatin sponge. A 1 cm gap was made in the middle portion of each of 10 consecutive tracheal cartilage rings in the canine cervix (control group, n = 3), then a gelatin sponge containing 12 microg of BMP-2 solution was implanted in the gap (12 microg group, n = 3). In another group (120 microg + P group, n = 3), the implanted gelatin sponge contained 120 microg of BMP-2 solution, and the gap was covered with periosteum. All of the control dogs developed saber sheath-type tracheomalacia, whereas tracheomalacia was not observed in the 12 microg and 120 microg + P groups. In the 12 microg group, fibrous cartilage was observed at the ends of the cartilage stumps. In the 120 microg + P group, newly formed bone and cartilage were observed to form a bridge between the cartilage stumps. The regeneration of cartilage or bone induced by the slow release of BMP-2 from a gelatin sponge might be useful for treatment of tracheomalacia.

For the regeneration of injured peripheral nerves, we have devised a PGA-tube that is composed of a tube of polyglycolic acid containing collagen sponge. This PGA-tube was applied clinically to reconstruct a peripheral nerve that had been resected during extended surgical resection of for intrapelvic recurrent rectal cancer. Four months after the surgical resection, the function of the left hip joint has improved remarkably, whereas the function had been lost just after the operation. It is suggested that the PGA-tube will be useful for regeneration of peripheral nerves that are resected during operation for intrapelvic malignancy.

We have established a method for in situ tissue engineering of the stomach in a canine model using an acellular collagen scaffold graft. The current study was conducted to evaluate the functional aspects of the tissue-engineered stomach wall. The anterior wall of the stomach in beagle dogs was replaced with a collagen sponge scaffold measuring 4 x 4 cm. At 16 weeks after implantation, the animals were sacrificed and the stomach specimens were evaluated immunohistochemically and physiologically. Regeneration of the proton pump and thin muscle layer, which are essential for mechanical and chemical digestion by the stomach, was observed in the tissue-engineered gastric tissue. However, acetylcholine-induced contraction was not observed in the tissue-engineered stomach wall. Although there is still room for improvement, the tissue-engineered stomach wall had a highly organized structure, and it is anticipated that this approach could eventually become an alternative for stomach reconstruction after gastrectomy.

Background/purpose: Circular myotomy often has been used to elongate the esophagus in end-to-end anastomosis for esophageal atresia with a long gap. Defects of the muscular layer often have led to the ballooning of the esophagus during the postoperative course. Recently, successful regeneration of the neo-esophagus using collagen sponge scaffold (CSS) for self-tissue regeneration has been reported. The authors applied this CSS to the muscular defect after circular myotomy in animal experiments., Methods: Myectomy or myotomy of the cervical or thoracic esophagus was performed in 11 piglets. CSS was applied to the amuscular site, which was 20 to 30 mm in length, to examine the regeneration of the muscular layer in 8 piglets (CSS group). The amuscular site was not wrapped by CSS in 3 piglets as a control (control group). Two months after operation, the animals were killed, and their operated esophagi were examined macroscopically and microscopically., Results: All 11 piglets increased in weight normally without any sign of dysphagia or vomiting. The amuscular site onto which CSS was implanted was covered with loose connective tissue, but muscle regeneration was not found microscopically. The amuscular site, which had been wrapped by CSS, was not distended in any of the 8 piglets when inflated, and ballooning was observed in all 3 of the control group., Conclusion: The results suggested that application of CSS to the site of circular myotomy for esophageal atresia with a long gap might not induce muscle regeneration but could prevent postoperative ballooning by inducing connective tissue growth., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

Purpose: It has been demonstrated that iodinated contrast medium (CM) causes the release of potassium into the intravascular spaces, resulting in an increase of serum potassium. The purpose of the present study was to assess the effect of mixing ratio on potassium release rates by using various mixing ratios with human blood in vitro., Materials and Methods: Fresh human blood from 52 patients was mixed in vitro with iodinated CM at ratios ranging from 10:2 (blood:CM) to 10:10. Potassium release rates were determined during 30 min of exposure to CM. The test solutions used consisted of 370 mgI/ml iopamidol, 320 mgI/ml meglumine/sodium ioxaglate, and 370 mgI/ml meglumine/sodium diatrizoate., Results: Potassium release rates increased gradually from ratios of 10:2 to 10:10. These changes were statistically significant. Among the three CM, diatrizoate induced the greatest potassium release, followed by iopamidol and then ioxaglate., Conclusion: Increasing mixing ratios caused an increase in potassium release rates.

Background: We have succeeded in regenerating the small intestine by the use of tissue engineering techniques. However, the regenerated intestine proved to lack the muscle layer, which is essential for functional peristalsis. To induce regeneration of the muscle layer, we focused on autologous mesenchymal stem cells (MSC) as a source of muscle tissue. The aim of this study was to investigate the effect of MSC seeding onto the collagen scaffold on induction of the muscle layer., Materials and Methods: We used six female beagle dogs. The small intestine was resected over a length of 5 cm and reconstructed by a collagen sponge graft in the same way as in our previous study. Autologous MSC derived from bone marrow (10(7) cells) were seeded onto the collagen sponge just before implantation. Animals were sacrificed at 2, 4, and 16 weeks after surgery, and specimens were examined histologically., Results: All six dogs survived until the scheduled time of sacrifice. At 4 weeks, regeneration of the intestine was observed at the reconstructed site. Cells positive for alpha-smooth muscle actin appeared on the scaffold in the MSC-seeded group. However, they disappeared by 16 weeks and only a thin muscle layer regenerated beneath the mucosal layer, although the regenerated mucosal layer covered the luminal surface of the regenerated intestine., Conclusions: MSC seeding induced a transient distribution of cells positive for alpha-smooth muscle actin on the scaffold, but did not induce regeneration of the muscle layer. Further investigation is necessary to achieve this aim., ((c)2001 Elsevier Science.)

The present investigation examined the key factors in the preparation of low-antigenic tracheal allografts by detergent treatment in dogs. In group 1 (n = 5), the grafts were treated by detergent at room temperature for 48 h and rinsed with running water until use. In group 2 (n = 4), detergent treatment was performed at 4 degrees C for 48 h, but the rinsing step was omitted. In group 3 (n = 6), the grafts were treated at 4 degrees C for 48 h, rinsed thoroughly with physiologic saline, and stored at 4 degrees C. The grafts were then used for tracheal replacement without immunosuppressant in dogs. The epithelium and mixed glands had been removed completely from the grafts in groups 1 and 2, and in five of the six grafts of group 3. In groups 1 and 2, cartilage viability appeared to have been lost and all animals died of airway stenosis within 38 days. In group 3, the chondrocytes were viable and all animals survived uneventfully. These results suggest that maintaining cartilage viability in a tracheal graft is necessary for successful immunosuppressant-free allotransplantation and, consequently, to maintain an open airway. The treatment temperature and sufficient rinsing are key factors for maintaining cartilage viability of grafts.

Recently, we have demonstrated canine peroneal nerve regeneration with functional recovery across an 80 mm gap using a polyglycolic acid (PGA) -collagen tube filled with laminin coated collagen fibers. In that study, the laminin coating was applied before a dehydrothermal (DHT) treatment designed to extend preservation of laminin in situ. To address concerns that the biological activity of laminin might consequently be reduced, the present investigation examined the influences of DHT crosslinking on the activity of laminin in terms of neural cell growth in vitro. DHT crosslinking was performed on collagen (type I or IV) spread on glass in three groups: (1) before coating, (2) after coating, and (3) both before and after coating. PC-12 cells were disseminated in each of the three groups. All three groups were cultured, and the number of cells were compared statistically. Cell growth achieved through application of laminin coating after DHT crosslinking was statistically greater than that achieved when laminin coating was performed before crosslinking. A reduction in laminin activity induced by DHT crosslinking was demonstrated. The optimal timing for the crosslinking of biornaterials treated with trophic factors such as-laminin should be examined in terms of the effects of crosslinking on the activity of the trophic factors.