Study On Bladder Repair With "Sandwich" Bionic Patch Constructed By Loading Adipose Tissue Extract To Electrospun Nanofibers

Part 1 Effects of Adipose Tissue Extract on Wound Healing and Growth and Differentiation of Rat Stem Cells and Endothelial CellsBackground and objective:Tissue engineering and regenerative medicine are important treatment to repair tissues and organs defect and realize their anatomical and functional regeneration.The survival and viability of stem cells and vascularization of new tissues are particularly important to achieve large-scale and long-distance tissue repair and replacement.Adipose tissue is not only an energy storage organ,but also a huge endocrine organ,which is rich in adipose-derived cytokines(also called adipocytokines)and has certain biological activity and physiological function.In this part,we intend to use a convenient method to prepare adipose tissue extract(ATE),to study its effects on stem cells and endothelial cells,and to explore its application value in tissue repair.Methods:ATE was extracted from subcutaneous adipose tissue of adult SD rats.Adipose-derived stem cells(ADSCs),bone marrow mesenchymal stem cells(BMSCs)and vascular endothelial cells(ECs)were extracted by reported methods.Two types of stem cells were cultured with ATE,and the effects of ATE on the proliferation of stem cells were detected by CCK-8.The expressions of Sox-2,Oct-4 and Nonag were detected to evaluate the effects of ATE on the pluripotent of stem cells.ECs were cultured with ATE.The effects of ATE on the proliferation,migration and tubularization of ECs were evaluated by CCK-8,scratch assay and tube formation assay,respectively.Furthermore,we used ATE as adjuvant and dissolved it with hyaluronic acid(HA)to prepared a kind of freeze-dried sponge dressing(ATE/HA)and coated it on the wound of rat skin,to observe its role in tissue repair.In addition,we used cytokine chip to analyze the components of ATE in order to explore and discuss the possible mechanism of its biological effects.Results:By using ATE,the proliferation of ADSCs and BMSCs was higher than that in control group,and the expression of stemness protein markers increased to a certain extent.The proliferation of ECs was also enhanced by ATE.The migration distance of ECs in ATE group was further than that in control group(85.92±15.52mm vs 35.33±24.85mm,p<0.05).In the transwell migration test,the number of migrating cells in ATE group was more than that in control group(19.8±4.8/hpf vs 1.2±0.8/hpf,p<0.05).In the tube formation assay,the average perimeter of tubules in ATE group was longer than that in control group(426.61±91.47mm vs 266.06±72.90mm,p<0.05)and the average area of tubules in ATE grpup was larger than that in control group(13314.29±6784.42mm~2 vs 5082.76±2433.00mm~2,p<0.05).In animal experiments,wound healing rate in ATE/HA group was faster than that in control group,and the expression of CD31 in ATE group was higher on the 10th day.In addition,cytokine chip was used to detect the components of ATE.The results showed that ATE contained many cytokines such as adiponectin,vascular endothelial growth factor,interleukin,brain-derived neurotrophic factor and so on.Conclusion:ATE contains many adipocytokines and has a variety of biological activities.It can promote the proliferation of stem cells and maintain the multipotency of stem cells.Moreover,ATE can promote the proliferation,migration and angiogenesis of ECs.In addition,ATE can promote the healing of skin wounds,which may be achieved by promoting angiogenesis.Therefore,ATE has the potential to be a kind of biological additive for tissue engineering repair and regeneration.Part 2 Adipose Tissue Extract Loaded on Electrospun Nanofibers to Prepare "Sandwich" Biopatch for Bladder RepairBackground and objective:Various congenital or acquired factors may lead to bladder injury or loss.Bladder tissue engineering is considered to be one of the ways to repair large defects of bladder and even replace bladder in the future.However,how to realize the functionalization of the neonatal bladder is an urgent problem to be solved in bladder tissue engineering.The key functional repair factors are considered to be the vascularization of endothelial cells and the migration and growth of urothelial cells and smooth muscle cells in neonatal artificial bladder.Electrospinning is one of the common methods to prepare nanofibers in tissue engineering.Drug-loaded nanofibers can not only achieve anatomical repair,but also load related drugs,proteins,cytokines and nanoparticles,which make the fibers to have certain biological activities to meet the needs of functional repair.Adipose tissue extract(ATE)is rich in adipocytokines and has the abilit ies to promote cell proliferation,migration and angiogenesis.Therefore,in this part of the study,we try to construct "sandwich" biopatches by loading ATE onto polylactic acid-polycaprolactone(PLCL)electrospun nanofiber membrane,in order to achieve the anatomical and functional repair of bladder.Methods: PLCL nanofibers were prepared by electrospinning.ATE and hyaluronic acid(HA)were mixed to prepare ATE/HA solution,coated it on PLCL fibers,and then lyophilized to prepare "sandwich" nanofibers biopatches(ATE/HA-PLCL).The characteristics of the patches were test by scanning electron microscopy,mechanical properties test and contact angle test.The histocompatibility was evaluated by subcutaneous transplantation in rats.Further,we prepared the rat bladder defect model,and repaired it with ATE/HA-PLCL patch,compared with PLCL patch.After 8 weeks,we performed micro-CT scan and urodynamic test to observe the bladder function.In addition,at 8 weeks after operation,HE staining and masson staining was used to evaluate the growth of urothelial cell and smooth muscle,and CD31 were used as immunohistochemical indicators to evaluate the effect of the patches on neovascularization.Results: The fiber diameter of ATE/HA-PLCL patch was 763 ± 31 nm,and the void ratio was 82.7 ± 2.4%.The ATE/HA layer had porous sponge structure,and the two layers are closely connected.The Young's modulus was 15.35 ± 1.47 MPa and the contact angle was 87.7 ± 1.8°.Four weeks after subcutaneous transplantation,there was no significant infiltration of CD45 and CD68 inflammatory cells.In the bladder repair experiment,by Micro-CT scan at 8 weeks after operation,no obvious bladder stones were found in ATE/HA-PLCL Group,while a small amount of stones attached to the bladder wall could be seen in control group.Urodynamic results showed that bladder capacity of ATE/HA-PLCL group was larger than that of PLCL group(1.53 ± 0.36 m L vs 1.16 ± 0.30 m L,p=0.07),and bladder compliance ATE/HA-PLCL group was also better than that of PLCL group(0.30 ± 0.10 m L/cm H2 O vs 0.18 ± 0.13 m L/cm H2 O,p<0.05).HE staining showed that the urothelium of the ATE/HA-PLCL group was multilayered and continuous.Masson staining showed that there were more myofibrillary tissues.And immunohistochemistry showed that there were more CD31-labeled neovascularization in the neobladder wall of ATE/HA-PLCL group.Conclusion: The ATE/HA-PLCL nanofiber patches have good mechanical properties and histocompatibility.It can promote the coverage of urothelium,the growth of smooth muscle and the formation of neovascular when applied to large-scale bladder repair.It can achieve the structural and functional repair of bladder to a certain extent.And it has certain research value and application potential in the field of bladder tissue engineering.