Improvement Of Implant Materials For Artificial Anal Sphincter

BackgroundFecal incontinence seriously affected the quality of patient's life. It happened with a number of diseases especially after rectal cancer operation. While the operation treats the diseases, it takes great inconvenience to the patients in the future.At present there is no effective treatment for incontinence. The clinical application of treatment includes reconstruction of their anal sphincter muscle transplant and artificial anal sphincter implantation. The former achieved a certain degree of self-anal. However, autologous transplantation is different from the anal sphincter muscle. It does not have the sensory function and can not take the initiative to control the bowel movements. Because of the limit of itself, its further improvement has great difficulties. Further more, the operation of both the gracilis muscle and other muscles is very difficult. It also has many postoperative complications and needs more time to recover. As a result, it is not conducive to the promotion.Implantation of artificial anal is more feasible in the treatment of incontinence. To make the effective control of defecation by artificial anus is possible through using of biomedical engineering. This is significantly important to the plight of fecal incontinence treatment.Traditionally, silica gel is often used as implanted artificial biological materials. Recently, some foreign researchers began to use polyurethane. Used with the latest nano-technology, we combined nano-hydroxyapatite with polyurethane which has the superior physical and chemical properties. On one hand, its biocompatibility greatly improved without causing systemic inflammatory response or immune rejection under the premise of artificial anal sphincter materials. On the other hand, the implant operation is much simple and may achieve the maintenance of anal pressure and self-restraint when combined with the icro-system technology. It can be produced and become industrialization once the technology is mature. The foreign developed artificial heart is a good example. So the research of artificial anal.sphincter has clinical value.ObjectiveStudy of nano-hydroxyapatite and polyurethane membrane composite and observe its in vitro and in vivo biocompatibility. In order to create a kind of artificial anal sphincter material that has excellent physical and chemical properties. It also has high biocompatibility to provide the best material choice for the establishment of intelligent artificial anus.Methods1. Preparation of nano-hydroxyapatite polyurethane composite membraneMedical polyurethane material was tailored into 65mm×80mm×2mm size. Using formaldehyde gas fumigation box disinfection to remove the surface dirt and then put it to place under the conditions of corona discharge of 15KV for 2 minutes. Then immersed it into the soluble amide which the concentration of 10%. Then it go through the various stages of polyurethane graft polymerization under 60℃.70℃concussion overnight and dry sealed.Put the pre-treated medical polyurethane under 37℃environment. A solution for the Ca solution, CaCl2/Tris-HCl, Ca10(P04)6(OH)2. B solution as the P solution, Na2HPO4, Ca10 (PO4) 6 (OH)2. Alternate immersion for each 10 seconds and remove to wash under the distilled water for 10 times and dried. Alternating 50-round altogether.2. The in vitro histocompatibility of nano-hydroxyapatite potyurethane composite membrane (nHA/PU)First Use inverted microscope to observation mouse fibroblast cell L929 culture with nHA/PU. nHA/PU was tailored into 10mm×10mm x 2mm size and put into the six-well plate after pre-wet-placed. Different concentrations of cells were inoculated-on the surface of the material to observe the growth of the cells above and around it.Second Measure the adhesion rate of the cells. nHA/PU was tailored into 5mm x 5mm x 2mm size and then pre-wet. Taking 24 pieces for the research group and 24 pieces for the negative group still 24 pieces for the positive group. Different concentrations of cells were inoculated on the surface of the material and then placed in an incubator. Then calculated the adherence rate of 8 holes each group at different time.Third We use scanning electron microscope (SEM) to observe the cells. nHA/ PU was tailored into 20mm x 20mm x 2mm size and placed in culture plate then inoculated the cells on its surface. And put the traditional silicone material as the control group. Finally, use SEM to observe the growth of the cells after three days.Fourth Detects cell cycle by flow cytometry (FCM). nHA/PU was tailored into 10mm×10mm×2mm size and then pre-wet. Taking 8 pieces for the research group and 8 pieces for the negative group still 8 pieces for the positive group.Put the cells culture with the material. And then detected different stage of the cells after a week and calculated index PI to observe the mater's impact on cell cycle. PI= [(S+G2/M)/(Go/G1+S+G2/M)] x 100%. Fifth CCK-8 Determination of cell growth curve. nHA/PU was tailored into 5mm×5mm×2mm size and then pre-wet. Taking 56 pieces for group A.56 plates with traditional silica gel for group B.56 plates with nHA/PU and medium for group C.56 plates with traditional silica gel and medium for group D. Put the cells culture in them. Choose eight holes of each group to test from day 2 to day 8 and then depicted the growth curve.Use SPSS 13.0 statistical software to analysis all the data and the experimental data was expressed by mean±standard (x±s). The rate of cell adhesion and cell cycle were analysis by one-way ANOVA. Use LSD method when doing mutual compare-between interclass and Tamhane's T2 if heterogeneity of variance. Use repeated measurement factor ANOVA to analysis the CCK-8 proliferation activity. Consider significant difference if P<0.05.3. The in vivo histocompatibility of nano-hydroxyapatite polyurethane composite membrane (nHA/PU)Select 18 healthy rats which body mass is about 200g. And then cut the nano-hydroxyapatite composite polyurethane into 5mm×2mm×5mm. The rats were randomly divided into PU group, experimental materials group and the control group. Each group has 6 rats. The rats were anesthetized and disinfection then cut for four incisions for each rat. The PU group was implanted with PU and the experimental-group was implanted with nHA/PU while the control group implanted with nothing. Choose one of the incisions in each group after different time and cut off the tissue around the implanted material. Then observe the inflammatory infiltration under optical microscope.Use SPSS 13.0 statistical software to analysis all the data and the experimental data was expressed by mean±standard (x±s). Use repeated measurement factor ANOVA to analysis the data. Use LSD method when doing mutual compare between interclass and Tamhane's T2 if heterogeneity of variance. Consider significant difference if P<0.05.Results1. Preparation of nano-hydroxyapatite polyurethane composite membraneWe access the polyurethane material which covered with nanometer-scale hydroxyapatite. X-ray diffraction and physical parameters test reports show the size achieve nano-level.2. The in vitro histocompatibility of nano-hydroxyapatite polyurethane composite membrane (nHA/PU)First Nano-hydroxyapatite polyurethane composite membrane was observed under an inverted microscope. Cells around the edge of nHA/PU materials are increasing over time.Second Cell adhesion rate of cells in the control group, experimental group and traditional materials after 1 hour were 58.6%,45.4% and 35.8%. Pairwise comparison among the three groups shows the differences were statistically significant (P<0.05). But the cell adhesion rate difference is not statistically significant 2 hours and 4 hours after inoculation.Third Under scanning electron microscope L929 cells in the traditional materials and nHA/PU surface can grow. The former cells were spindle-shaped with a small amount of pseudopods protruding slightly between cells connected while the latter cells were "sea star" growth. They are many processes on the surface and through the processes linked with surrounding cells. A large number of pseudopodia and microvilli adhered material can be seen. This indicates that cells are in good condition. Fourth The flow cytometry showed that the control group, experiment group and the traditional materials of cell are all normal diploid cells and no aneuploid cells to form. Both traditional material and nHA/PU composites have little to do with mouse fibroblast cell growth cycle.Fifth CCK-8 was used to detect the cell proliferation over nHA/PU material. From the growth curve which is draught from the OD value of cells activity we can see the cells maintain a certain degree of division and proliferation rate in both nHA/PU group and traditional after inoculation. There is significantly different (P=0.000) in each group at different time. And the absorbance values increased with time. There is significantly different (P=0.014) at the same time between the two groups. The experimental group was higher than traditional materials group. And the interaction effect between the groups and time was not obvious. All appear "S"-shaped increments(P= 0.882).3. The in vivo histocompatibility of nano-hydroxyapatite polyurethane composite membrane (nHA/PU)In each group which were observed there is a certain inflammatory response but gradually improved over time and almost disappear after 12 weeks. Inflammatory cells and capillary counting results showed that the number of lymphocytes and neutrophils in each group at different time has significantly difference, both inflammatory cells and capillary number is different from the control group. And the polyurethane group has the most while the control group has the least. Among these three groups. The control group reached to the peak after 1 week and then gradually decreased, while the urethane group and composite materials group reaching a peak after 4 weeks and then gradually decreased. Both reduced to a minimum after 12 weeks.Conclusions1. Combine high compatibility of nano hydroxyapatite and medical polyurethane together to product artificial anal sphincter implant is feasible.2. Nano-hydroxyapatite polyurethane composite membrane and mouse fibroblast L929 has good histocompatibility in vitro and found no significant rejection and toxicity.3. Nano-hydroxyapatite polyurethane composite membrane has good biocompatibility in rats and did not cause rejection after implantation. It also has a good combination with the surrounding tissue.Incontinence is caused by various reasons. Dysfunction of anal self-control is caused by multiple factors involved in clinical symptoms. The treatment of this disease is difficult to functional reconstruction of the anus while the artificial anal sphincter implantation is the hope of treatment. The ideal of artificial anal-sphincter implant material production is the key. We believe intelligent artificial anus in situ development will continue to improve and clinical treatment of incontinence provides an ideal solution. So the research of artificial anal sphincter has clinical value.