| Objective: The degumming method is used to prepare the silk protein membrane and the solvent casting method is used to prepare the polylactic acid membrane.It’s hoped to find a biological material which is better for the repair of colonic wall defect to lay a foundation for the treatment of colonic defect after physical properties and biological effects of above-mentioned membranes are discussed and their effects on colon repair in rabbits are compared.Methods:(1)Bombyx mori cocoons were prepared by degumming,lithium bromide solution dissolution and other methods for preparing silkworm protein membranes;dissolving medical polylactic acid materials in dichloromethane and other methods to prepare polylactic acid membranes.The physical properties of the two films were evaluated by scanning electron microscopy to examine the morphology of the film,as well as the swelling rate test and tensile test.The biocompatibility of Silk protein membrane and Polylactic acid membrane was evaluated by CCK8 experiment and cell death staining.(2)36 healthy New Zealand Rabbits(weight: about(2±0.2)kg)are chosen.They were randomly divided into 3 groups.Group A(n=6),namely blank control group;Group B(n=15),namely silk protein membrane repair group;Group C(n=15),namely polylactic acid membrane repair group.After establishing a full-thickness(1×1cm)perforation model of rabbits in groups B and C,the biofilm and the colon wall were sutured by continuous suture.Periodically observe the experimental rabbits of each group and record the body weight,feed intake,C-reactive protein and white blood cell value of each group of rabbits.The rabbits in each group were treated with euthanasia at 1、5、9 and 13 weeks after surgery to observe the infection of the abdominal cavity,the healing of serous and mucosal layers,and adhesion.Samples of the repaired area were collected and fixed with formaldehyde,followed by HE staining and Masson staining.(3)IHC staining of the collected samples was used to observe the expression of colonic smooth muscle actin(α-SMA).Results:(1)a.Scanning electron microscope(SEM)showed that the surface of Silk protein membrane was smooth,and the surface of Polylactic acid membrane showed radial protrusions.The test result of the swelling rate showed that the silk protein membrane were 33.5 ± 0.47% and the polylactic acid membrane were 25.9 ± 0.35% at13 minutes.The difference was significant(P﹤0.001).Under dry conditions,the stress of the silk protein membrane can reach 5.57 ± 0.073 MPa,and the stress of the polylactic acid membrane can reach 7.45 ± 0.056 MPa.The stress between the two is significantly different(P﹤0.05).b.72 hours after incubation of L929 cells with Silk protein membrane extract and Polylactic acid membrane extract,all cells showed normal fusiform,cell death staining showed that both groups emitted green fluorescent signal,and very few dead cells showed red fluorescence.CCK-8 experimental results,the cell survival rates detected at 24 h,48h,72 h,the survival rates of L929 cells treated with Silk protein membrane extract were 78.73%、92.1%、95.6%、and the survival of L929 cells treated with Polylactic acid membrane extract The rates were 77.6% 、92.33%、96.4% and there was no statistical significance between the two(P > 0.05).(2)a.One postoperative group B and C each died with a mortality rate of 6.6%.Within 20 days after operation,2 rabbits in group B had poor mental state and poor appetite.The remaining test rabbits were asymptomatic,diarrhea,wound infection,fecal incontinence and other symptoms.The feed intake of rabbits in groups B and C returned to the level of group A about 15 days after surgery.Within 1 week after operation,the weight of rabbits in groups B and C decreased.The weight of group B returned to the preoperative level about 30 days after the operation,and the weight of group C returned to the preoperative level about 21 days after the operation.b.The CRP value of group B increased faster than that of group C within 3 days after operation.The CRP value of group B and group C reached the peak at 5 days after operation,and then gradually decreased.The CRP value of group B and group C returned to within 10mg/L at 21 days At 27 days,the CRP values of groups B and C returned to the level of group A,and the comparison of groups B and C with group A(P﹤0.01).c.The WBC value of group B rises faster than that of group C within 3 days after operation.Group B reaches its peak at 3 days,and reaches the peak at 5 days in group C,and then gradually decreases.The decrease rate of group B is faster than that of group C between 5 and 13 days.Large,the WBC values of both groups were close to group A after 25 days,and there was no statistical significance between the two phases of B and C(P>0.05).The comparison between the two groups of B and C and group A(P ﹤ 0.01).d.Postoperative dissection observation,except for 1 death caused by intestinal obstruction caused by grade Ⅱ adhesion in group B and 1 death caused by abdominal abscess in group C,the remaining test rabbits did not die,and there were no leakage,no stenosis,and no intra-abdominal cavity at the colon repair site Complications such as abscesses.e.HE and Masson staining results,comparing groups B and C with group A.Inflammatory cell infiltration was observed in groups B and C at 1 week postoperatively;inflammatory cell infiltration in subserosal layer of group B and C was shown at 5weeks postoperatively,collagen fibers proliferated and accompanied with neovascularization;The mucosal layers of group C all recovered,and the inflammatory cells infiltrated beneath the serous layer of group B;13 weeks after operation,the muscular layer of group B showed incomplete recovery.The structure of each layer of colon in group C recovered,and the degree of recovery was close to that of group A.f.IHC evaluation showed that the B and C groups were compared with the A group,and a very small amount of α-SMA positive expression was shown in the B and C groups 1week after operation;the round and linear strong expression(α-SMA)More than group B;9-13 weeks after surgery showed that the smooth muscle layer reconstructed in group C expressed higher expression of α-SMA contractile protein than group B,and its expression maturity was similar to that observed in group A.Conclusions:(1)The in vitro test shows that these two membranes can support proliferation and adhesion of L929 cells and are free from cytotoxicity,but the stress of the polylactic acid membrane is stronger than that of the silk protein membrane;(2)The in vivo test shows that they can effectively repair the colonic defect in rabbits,but the polylactic acid membrane repair group has a better effect in terms of tissue adhesion resistance,tissue defect healing speed and enhancing the regeneration of colonic smooth muscles.To sum up,the polyemulsion membrane has a better effect when it is used for rabbit colon defect repair. |
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