| The regenerative ability of bile duct after injury was limited; surgery is often accompanied by severe biliary complications. With the development of tissue engineering and biodegradable semi-synthetic artificial bile duct, new hopes were brought to solve this problem.In our study, we utilized a new artificial bile duct patch and tube to repair the bile duct wall defect and segmental bile duct defect. We evaluated the regeneration of epithelium cells, accessory glands, vessels, smooth muscle and scar formation after the defect was repaired by collagen scaffold and collagen scaffold loading with CBD-bFGF. The purpose of current study was to evaluate the healing process and the prevention of postoperative biliary complications after using this new material.Part One:Experimental study on bile duct wall regeneration in pigs using artificial bile duct patchObjective:To evaluate the effect of collagen scaffold patch loaded with CBD-bFGF in the regeneration of bile duct wall in pigs.Methods:The pore size and porosity of collagen material were detected according to the reference. Then the degradation rate of collagen material was evaluated by bile soaking experiment in vitro. Totally 38 Chinese experimental pigs were assigned to three group, including Collagen/PBS group (bile duct wall defect was patched by a collagen membrane), Collagen/CBD-bFGF group (bile duct wall defect was patched by a collagen membrane loaded with CBD-bFGF) and normal control group (pigs were killed to obtain normal bile duct by time).4 weeks,8weeks and 12 weeks postoperatively, liver function and blood routine were detected and compared. The cholangiography was performed under fluoroscopic guidance by cholecystostomy to evaluate if there was any evidence of lithogenesis or stricture formation at 12 weeks. The neo-bile ducts were obtained to detect the burst pressure after general observation. HE staining, Masson staining and Immunohistochemical staining of CK19, vWF, Desmin and α-SMA were detected.2 pigs in Collagen/CBD-bFGF group were fed to 24 weeks and evaluated the effect of long term repair.Results:The average pore size is 186.65±45.37μm and average porosity is 84.31±6.5%. The collagen material can maintain its form around 3 weeks in bile. Animals in both groups appeared healthy at the time of observation. The GGT and TBA in Collagen/CBD-bFGF group were significantly lower than in Collagen/PBS group. Blood routine test and cholangiography did not show abnormality in both groups. The burst pressure was satisfied after 8 weeks in both groups. Masson staining showed collagen structure in Collagen/CBD-bFGF group were more regular to the normal control group than collagen scaffold/PBS group. The amount of collagen deposition in Collagen/CBD-bFGF group was decreasing significantly by time. Immunohistochemical staining showed a more quickly epithelialization, gland regeneration, vascularization, and smooth muscle regeneration rate in Collagen/CBD-bFGF group compared to Collagen/PBS group. The amount of Myofibroblast was significantly lower in Collagen/CBD-bFGF group than in Collagen/PBS group. There was a positive correlation between accessory glands counting and MVD counting and at 4 weeks postoperatively, the accessory glands counting were significantly higher in Collagen/CBD-bFGF group than in Collagen/PBS group. There were no stricture formation or lithogenesis in Collagen/CBD-bFGF group at 24 weeks, the structure of the bile duct wall were similar to normal bile duct.Conclusion:1. Collagen scaffold patch could complete the reconstruction of a bile duct wall defect (2 cm in length and 1/3-2/3 of the bile duct circumference in width). 2. The collagen scaffold loaded with CBD-bFGF could effectively improve the regeneration of epithelial cells, glands, vessels and smooth muscles, and low the risk of bile duct stricture.3. CBD-bFGF could promote the hyperplasia of accessory glands by promoting the regeneration of blood vessel formation.Part Two:Experimental study on segmental bile duct regeneration in pigs using artificial bile duct tubeObjective:To evaluate two collagen scaffolds with different degradation rate in the regeneration of segmental bile duct in pigs. To evaluate the effect of collagen scaffold patch loaded with CBD-bFGF in the regeneration of segmental bile duct in pigs.Methods:The density, pore size and porosity of two collagen materials were detected and compared. Then the degradation rate of two collagen material was evaluated by bile soaking experiment in vitro. Firstly,8 Chinese experimental pigs were assigned to two group, including Collagen A group (the same scaffold used in part one) and Collagen B group (the scaffold was improved based on the same scaffold used in part one), these pigs were fed for 4 weeks to evaluate survival rate, liver function, blood routine tests and HE staining. The scaffold with better results was selected to load with CBD-bFGF. Then,27 Chinese experimental pigs were assigned to three group, including Collagen/PBS group (segmental bile duct defect was repaired by a selected collagen scaffold tube), Collagen/CBD-bFGF group (segmental bile duct defect was repaired by a selected collagen scaffold tube loaded with CBD-bFGF) and normal control group (pigs were killed to obtain normal bile duct by time).4 weeks,8weeks and 12 weeks postoperatively, the complication rate, liver function and blood routine test were detected and compared. The cholangiography was performed under fluoroscopic guidance by cholecystostomy to evaluate if there was any evidence of lithogenesis or stricture formation at 12 weeks. HE staining, Masson staining and Immunohistochemical staining of CK19, Ki67, vWF, Desmin and a-SMA were detected to evaluate the repair effect.Results:The density is significantly higher in collagen scaffold B than in collagen scaffold A but there was no significant different in average pore size and average porosity between the two collagen materials with different degradation rate. After bile soaking, the degradation rate was slower in collagen scaffold B than in collagen scaffold A. The animal experiment showed all pigs died using collagen scaffold A because of bile leakage, infection, gastrointestinal hemorrhage and malnutrition. After using collagen scaffold B, all pigs survived to 4 weeks. The animal experiment of collagen scaffold B loaded with CBD-bFGF showed:the rate of biliary stricture in Collagen/CBD-bFGF group were significantly lower than in Collagen/PBS group, but there were no difference in the mortality rate and infection rate between the two group. After loaded with CBD-bFGF, the materials collapsed after the degradation, and biliary stricture and biliary infection took place in some experimental animals. These were result in impaired liver function and malnutrition at 4 and 8 weeks. The animals survived to 12 weeks in Collagen/CBD-bFGF group were better than in Collagen/PBS group in liver function and nutrition. Cholangiography show stricture formation in Collagen/PBS group in 12 weeks, while there was no stricture in Collagen/CBD-bFGF group in 12 weeks. Pathologically, HE staining showed the area of scar and inflammatory cells in Collagen/CBD-bFGF group was less than in Collagen/PBS group. Masson staining showed collagen structure in inflammatory cells group was regular and the thickness of bile duct wall were less than Collagen/PBS group. Immunohistochemical staining showed a more quickly epithelialization, gland regeneration, vascularization, and smooth muscle regeneration rate in Collagen/CBD-bFGF group compared to Collagen/PBS group. The amount of Myofibroblast was significantly lower in Collagen/CBD-bFGF group than in Collagen/PBS group.Conclusion:1. Collagen scaffold tube could only induce scar healing process in segmental bile duct defect around 2cm.2. The collagen scaffold loaded with CBD-bFGF could effectively improve the regeneration of epithelial cells, glands, vessels and smooth muscles, and low expression of MFB, but accompanied with the problem of quick material degradation rate and collapse to form stricture, thus needed to be further improved. |
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