| Part1Objective (1) To clarify the dynamic changes of the binding lines during the healing process of intestinal anastomoses via the sutureless manners using intraluminal biofragmentable stents;(2) To verify the safety and feasibility of the intestinal anastomosis using the intraluminal biofragmentable stent at an extreme approximation of "mucosa-to-serosa";(3) To further prove the isolation effect from the intestinal contents and the biomechanical forces within the early period of the intestinal anastomoses using the intraluminal biofragmentable stents, which provides the theoretical support for the subsequent in vitro experiments.Methods Taking Bama miniature pigs as the experimental subjects, we performed the "mucosa-to-serosa" anastomoses of the small intestine and colon respectively, using hand-sewn manner and the intraluminal biofragmentable stents manner. At the postoperative days (POD)3,7,14,30, and90, the postoperative mortality and complications of each group were recorded; the anastomotic healing, the stent shape, and the position of binding lines were observed; the anastomotic bursting pressure and hydroxyproline contents were determined; anastomotic tissues were histologically analyzed under the hematoxylin-eosin (HE) staining, Masson staining, the immunohistochemical staining of anti-TGF-β1, anti-b-FGF, anti-α-SMA and anti-CD31; transmission electron microscopy was also used; TGF-β1, b-FGF and of α-SMA mRNA expressions were evaluated as well via real-time reverse transcription polymerase chain reactions (RT-PCR). In addition, the isolation effect from the intestinal contents and the biomechanical forces within the early period of the intestinal anastomoses using the intraluminal biofragmentable stents was proved by the methods of continuous perfusion, intestinal contrast imaging under X-rays and bursting pressure measurement.Results (1) The binding lines represented a dynamic process of slow-moving from the serosa to the mucosa, no matter whether the enteroenterostomy or colocolostomy. During POD3-7, the binding lines located at the muscularis approximately, POD7-14at the submucosa, POD14-30at the mucosa or inside the intestine. Though they caused histological granulomatous changes as foreign bodies, the binding lines did not cause intestinal abscess or penetrating injuries. During the movements, the lateral structure healed soon once the binding lines reached the inner layers, as in the seton therapy.(2) The intestinal anastomosis using the intraluminal biofragmentable stent is safe and feasible, even at an extreme approximation of "mucosa-to-serosa", no mortality occurred. Macroscopically compared to the hand-sewn manner, the healing scar was smaller, and no postoperative anastomotic stricture and related intestinal obstruction occurred. The bursting pressure of the stent group on POD3and POD7were significantly higher than the hand-sewn group (p<0.05); The bursting sites were also different:in the stent group, the bursting sites were on the normal intestinal wall underneath or outside the binding lines, whereas in the hand-sewn group, the bursting sites were just on the anastomoses or the needle holes. The bursting pressure on POD14-90were comparable between the two groups (p>0.05). The hydroxyproline contents of the small intestine anastomoses in the stent group were higher than the hand-sewn group on POD7and POD14(p<0.05), and there were no differences in the rest of the time (p>0.05); whereas in the colon anastomoses, the hydroxyproline contents in the stent group were higher than the hand-sewn group only on POD14(p <0.05). Microscopically, the inflammatory response in the stent group subsided faster, the cell numbers and function of fibroblasts peaked earlier, and the scar maturity was higher, compared to the hand-sewn group. The TGF-β1and b-FGF mRNA expressions in the stent group were lower than the hand-sewn group; whereas the a-SMA expression was higher in the early postoperative period and lower in the late period. Dynamic changes of the fibroblast cell number, function and the collagen secretion were observed in the transmission electron microscopy.(3) The completely isolation effect from the intestinal contents and the biomechanical forces within POD14of the intestinal anastomoses using the intraluminal biofragmentable stents was proved by the methods of continuous perfusion, intestinal contrast imaging under X-rays and bursting pressure measurement.Conclusion (1) The binding lines represents a dynamic change of "chronic cutting-and-healing", like the seton therapy, in the healing process of intestinal anastomoses using the biodegradable stents. They complete the process from the outside to the inside of the intestine between14-30days, and they cause little damages to the intestinal wall.(2) During the early period of the intestinal anastomoses using the intraluminal biofragmentable stents, the stents and the binding manner can completely isolate the anastomoses from the intestinal contents and the biomechanical forces.(3) The intestinal anastomosis using the intraluminal biofragmentable stent is safe and feasible, even in the case of an extreme "mucosa-to-serosa" approximation.(4) Compared to the hand-sewn group, the isolation due to the biodegradable stents creates an environment which is benefit for the intestine healing and promotes the healing of the anastomoses. Part2Objective To study the impact of three biomechanical forces, intraluminal pressure, strain, and shear respectively, on the intestinal healing in vitro.Methods Taking Caco-2cells and the primary cultured human intestinal fibroblasts (PHIF cells) as experimental subjects, the static pressure as0mmHg,20mmHg,60mmHg, static strain as0%,20%,60%stretch, and shear force generated by the medium velocity as0cm/s,4cm/s,8cm/s,16cm/s, were applied. After24hours, AnnexinV-FITC/PI double staining method to detect apoptosis (flow cytometry and confocal laser scanning microscope); Rhodamine-Phalloidin/DAPI double staining (laser scanning confocal microscope) to determine the cell viability; wound healing assay to detect the cell migration; enzyme-linked immunosorbent assay (ELISA) of TGF-β1, EGF, a-SMA, Col-1, and PⅢNP expression; and Western Blot detection of TGF-β1and EGF downstream pathways were performed respectively.Results Under the different biomechanical forces, the apoptotic rates of Caco-2cells were increased with the forces levels, whereas apoptosis of the PHIF cells was relatively not significant, though the cell viability decreased; in the wound healing assay, the wound closure rates of PHIF cells decreased with the forces levels, suggesting the down-regulated migration capability; the TGF-β1, α-SMA, Col-1, and PIIINP expressions were increased with the forces levels via ELISA, whereas the EGF expression did not up-regulate significantly or even slightly decreased; the activated TGF-β1/Smad pathway was detected by Western Blot assay, whereas the EGF pathway was not activated.Conclusion The biomechanical forces are not conducive to the intestinal anastomotic healing, therefore the isolation due to the biodegradable stents creates an environment which is benefit for the intestine healing and promotes the healing of the anastomoses.
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