Experimental Study Of Biological Evaluation Of A Novel Pelvic Floor Repair Patch

Objectives:To investigate the biological properties of porcine urinary bladder matrix (UBM) fixedwith genipin in vitro, evaluate biocompatibility and biomechanical properties of graftsimplanted in rabbit abdomen and vagina, so as to provide the experimental basis for clinicalapplication of novel pelvic floor reconstruction patch.Methods:1. Cellular components of porcine bladder were removed by detergent-enzymaticmethod. The acellular porcine bladder was crosslinked with0.625%genipin for72h, thenexamined the morphology with hematoxylin and eosin staining and scanning electronmicroscope. Properties of the crosslinked acellular matrix were evaluated by tissuethickness measurement, water absorption，in vitro enzymatic degradation test and thebiomechanical tests. The cytotoxicity of the materials was evaluated in vitro by MTT assayusing a murine fibroblast cell line L-929.2. Animals were randomly divided into6separate groups,there were15rabbits ineach group. One of the following materials was implanted in the abdominal of therabbits:(A)UBM,(B)cross-linked UBM,(C)polypropylene(PP) mesh,(D)compound cross-linkedUBM,(E)compound UBM. The rabbits were sacrificed at1,2,4,8,12weeks after surgery,and part of the samples were subjected to hematoxylin-eosin (H&E) staining to histologicaltest, RT-PCR was used to detect the dynamic changes of IFN-γ, IL-2, IL-4and IL-10mRNA level. The sham operated group underwent similar procedures, but with noimplantation.3. A total of40adult New Zealand White female breeder rabbits were implanted withPP mesh (n=20) orgenipin-crosslinked UBM (n=20) in the vagina and abdomen. Graftswere harvested12weeks later and were processed for histology to evaluate the hostinflammatory response and degree of tissue incorporation within the grafts,and underwent mechanical properties testing.Results:1. The genipin-crosslinked porcine urinary bladder matrix was dark blue, with verywell preserved collagen fiber. Cosslinked tissues had strong hydrophilicity and goodmechanical properties, the degradation ratio was only2%at28days after collagenasetreatment. The urinary bladder matrix was degradated completely at7days aftercollagenase treatment. The cytotoxicity of the materials was scored as grade0to1.2. D、 E group were associated with erosion response, E group had the mostinflammation score, compared to other group，the difference of which was considered asstatistically significant (P <0.05); neovascularization, fibroblast proliferation score was thehighest in the polypropylene group,compared to the other five groups,the difference wasstatistically significant (P <0.05), there were significant statistical difference in fibroblastsproliferation compared each implantation group to sham group(P＜0.05).The degradationspeed was the fastest in A group, which was complete degradation after12weeks ofsurgery,there weren’t degradation in B group. IFN-γ, IL-2, IL-4and IL-10mRNAexpression level of B group at each time point were similar with sham group.3. Vaginal PP was associated with erosion reaction(50%), Abdominal PP andcross-linking UBM group had no erosion phenomenon.Vaginal PP (33%) were missing, allabdominal PP were identified at necropsy, abdominal crosslinked UBM (6%)were missing,vaginal crosslinked UBM (53%) were missing. All the patchs had adhesion with rectusabdominis or vaginal mucosa, and had various degrees shrinkage, PP were more obvious.4. Cross-linking UBM induced,a milder chronic inflammation response comparisonwith polypropylene, had lower scores for inflammation (P=0.021),and had higher scores forneovascularization (P=0.000) and fibroblastic proliferation (P=0.008) than PP. Vaginal pphad higher scores for inflammation (P=0.001) than abdominal PP, and they were similar toneovascularization and fibroblastic proliferation(P>0.05).5. After implantation,the mechanical properties of UBM was not weakened,PP hadincreased tensile strength and elastic modulus. PP had higher scores for tensile strength,relative elongation at failure, break strength than crosslinked UBM after implantation inabdomen(P＜0.05); PP had higher scores for tensile strength, break strength thancrosslinked UBM after implantation in vagina(P＜0.05), elastic modulus difference was not significant statistically (P>0.05).Conclusions:1. The Genipin-fixed UBM has the ability of good hydrophilia, cytocompatibilityand resistance to enzymatic degradation. It can be used as an ideal tissue engineeringmaterial for the reconstruction of female pelvis.2. The cross-linked UBM had slow degradation, mild inflammatory response, and lowimmunogenicity.Genipin-crosslinked UBM has better compatibility, more easily integrationwith vagina, mechanical performance is not decreased in vivo. It may be a promisingmaterial for pelvic reconstruction.