Application Of Hyaluronic Acid Coaxial Nano-electrospinning And Adipose Derived Stem Cells In Bladder Tissue Engineering

Congenital malformations or acquired diseases such as:infection,malignant tumor,trauma,often cause most or even whole loss of the bladder wall.The clinical routine treatment is using gastrointestinal line to establish new bladder or urinary tract output.However,the complications caused by the secretion and absorption of gastrointestinal mucosa have become the major obstacle in clinical application.With the development of regenerative medicine,bladder tissue engineering has attracted researchers' attention.There are two ways to repair the defect of the bladder by tissue engineering:single scaffold and seed cell composite scaffold.Single biological material technology sometimes had complications such as:stone,graft contracture,poor angiogenessis and inadequate vascular smooth muscle and so on.In order to overcome this problem,the composite seed cells with scaffolds is widely adopted.Adipose derived stem cells(ASCs)derived from mature adipose tissue in vivo,which could be obtained in large quantities,attracted people's attention.ASCs was a new kind of "seeding cell" from adult stem cells.Some scholars had matured ASCs differentiate into smooth muscle cells(myogenically differentiated adipose derived stem cells,MD-ASCs),for the study of bladder regeneration.However we found the differentiation process was too long to clinical application.So we propose to optimize the selection of seed cells by improving the MD-ASCs differentiation.Polymer scaffolds and electrospinning methods are the most important parts of tissue engineering.Electrospinning of polymer materials in electrostatic field can be stretched diameter of fibers of the nano-level,it can greatly improve the contact area of scaffold and cell,which is benefit to cellular adhesion and proliferation.Compared with acellular biomaterials,electrospinning has the following advantages:controllability of the fiber surface shape and internal structure,encapsulating drug or growth factor into fiber,guiding the direction of orientation of fiber cell growth,etc.However,the biocompatibility of polymer materials is still a problem to be solved.At present,polylactic acid,polycaprolactone,chitosan,chitosan are applicate,but their bio-function is poor.Using tissue engineering scaffolds for small area bladder repair could observe a good effect on tissue repair;however the repair area was more than 50%,although the urothelial cell regeneration,bladder smooth muscle regeneration was poor.In the center region of neo-bladder,low microvessel density,disorder and less smooth muscle could be observed.Hyaluronic acid(HA)is an important extracellular matrix of bladder smooth muscle cells,HA can promote the proliferation and migration of smooth muscle cells,formation of blood vessels,and reduce scar formation during wound healing.Meanwhile,the aqueous solution of HA is a viscoelastic fluid,which is filled in the space between the cells and collagen fibers and covered in some epidermal tissues.The main function of HA in the body is to protect and lubricate the cell,regulate the movement of the cell on the viscoelastic matrix,stabilize the collagen network structure and protect from mechanical damage.We proposed using electrospinning technology to construct a 3D scaffold,which is composed of HA nanofibers,could simulate the extracellular matrix to promoted cell growth and angiogenesis.In this study,we associated E2 with MD-ASCs differentiate process,and fond that E2 led to modulation of the MD-ASCs phenotype toward a concentrated type with smooth muscle-inductive medium.At the same time,the "core-shell" structure of HA-PLCL nano-scaffold was fabricated by coaxial electrospinning technique.We transplanted MD-ASCs onto HA-PLCL scaffolds,and then made the composite to explore the feasibility of bladder repair.Part One:The Effect of Proliferation and Myogenic Differentiation of Association 17-? Estradiol with Adipose-Derived Stem CellsObjective:To study the effects of 17?-estradiol on proliferation and the process of myogenic differentiation of rat adipose-derived stem cells in vitro.Methods:SD rat inguinal adipose tissue was digested by collagenase for isolate adipose derived stem cells and cultured in vitro to the third generation,Adipogenic differentiation and osteogenic differentiationthe were induced to determine the multi-directional differentiation potential,stem cell surface antigens(CD45,CD73,CD90)were identified Flow cytometry.Estrogen was diluted into different concentrations(10-7,10-8,10-9,10-10,10-11M)by DMEM culture medium,which was used for the proliferation of ADSCs in vitro.MTS method was used to evaluate the effect of estrogen on proliferation of ADSCs in vitro.The most suitable concentration was chosen for investigating the effect on the process of myogenic differentiation,adipose-derived stem cells were induced by smooth muscle inductive medium with or without estrogen by 2 and 4 weeks.Immunofluorescence,real-time quantitative polymerase chain reaction(Real-time PCR)and Western blot(Western Blot)method was used to detect early myogenic markers(alpha-SMA),middle myogenic marker(SM22,Calponin)and late myogenic markers(MHC)expression.Results:Estrogen cloud effectively promote the proliferation of ADSC in the concentration range of 10-7-10-10M in the male rats and 10"7-10-11M in the female rats.10-9M estrogen could promote the proliferation more effective than others concentration groups in male or female rats.By 2 weeks matured,early signs of smooth muscle differentiation in estrogen group cells(alpha-SMA),middle differentiation markers(SM22,Calponin),late differentiation markers(MHC)expression were higher than those in the non-estrogen group(P<0.05).Sequentially,the expressed level of early and middle myogenic marker was same,while the late marker expression of estrogen group was higher than that of non-hormone group by 4 weeks differentiation(P<0.05).Conclusion:1.Estrogen incorporation could enhance the proliferation of ASCs in vitro,and the most optimal concentration was 10-9 M.2.Estrogen also led to modulation of the myogenically differentiated adipose derived stem cells(MD-ASCs)phenotype toward a concentrated type with smooth muscle-inductive medium.The expression of early,mid,and late-stage contractile markers in MD-ASCs was enhanced by E2 during the different differentiation stages.Part Two:Biocompatibility Study of HA-PLCL Coaxial and PLCL ElectrospunObjective:To study the characterization and physical and chemical properties of HA-PLCL co axial electrospun,and to investigate the biocompatibility and feasibility of repairing the bladder defect by HA-PLCL composite.Methods:1.characterization:using coaxial electrospinning technology to produce shell is hyaluronic acid(HA),nuclear layer is polylactic acid/polycaprolactone(PLCL)coaxial electrospinning,meanwhile producting PLCL blend electrospun and shell is PLCL core layer is HA electrospun as control groups.The surface and internal characteristics of electrospinning materials were observed by SEM and TEM,the hydrophilicity was detected by hydrophilic contact angle measurement method and cross section observation of swelling,testing the mechanical properties of universal tensile tester,degradation rates was measured by weight losing within continuous 7 days PBS soaking in vitro for degradation rate.2.Detecting cell compatibility of materials by seeding cells morphology using optical microscope,confocal microscope,scanning electron microscope;the proliferation and adhesion of cells in the materials was detected by MTT and high speed centrifugation method;clone ring culturing cells was observed in the material for the migration of cells;immunohistochemical staining of smooth muscular cells features in scaffold composite to verify the myogenic maintenance in scaffolds.3.Histocompatibility and bladder repair:the material was planted in the subcutaneous of rats for 4 weeks,scaffolds were taken for histological analysis.The model of bladder defect was made by removing the top of the bladder,and then scaffold composite was sutured to the defect,estimating neo-bladder by urodynamic in 4 and 8 weeks post-operation.Results:1.Characterization of scaffolds:By SEM observation,HA-PLCL and PLCL had the neat and smooth surface,uniform fiber diameter.PLCL-HA spinning jet beads is serious.By TEM observation,HA-PLCL coaxial electrospinning had the shell-core structure,PLCL spinning internal structure symmetry is single,the internal structure of the PLCL-HA to form a similar projectile like structure,not uniform tensile.PLCL,HA-PLC and PLCL-HA diameter were about:671 ±34nm,781 ± 46nm,411 ±102nm.The outer layer thickness of HA-PLCL electrospinning is about 252± 56nm,the inner diameter is about 277±34nm.The production efficiency of PLCL-HA is much less than that of the other two groups.The mechanical properties and degradation rates in vitro of HA-PLCL and PLCL were similar.Hydrophilicity of HA-PLCL was higher than PLCL after 3 days soaking in PBS,and swelling and delamination occurred inside the HA-PLCL.2.Cellular compatibility of scaffolds:cells could grew well in two groups,but better cell spreading shape and filarpseudopodia was showed earlier in HA-PLCL group.The cloning ring culturing showed that the cells migrated well on HA-PLCL.We observed that the proliferation of cells in HA-PLCL was faster than PLCL and so was about the cell adhesion.a-SMA and MHC staining were found the seeding cell can maintain complete myogenic features.3.Histocompatibility and bladder repair:HE staining and CD68 immunohistochemistry stain showed that the two groups of materials were maintained in the body for 4 weeks,and the cells could grow on the material.CD68 staining showed that the two groups of materials can cause mild macrophages.At 4 and 8 weeks after operation,the bladder repaired animals survived well without serious complications.Urodynamic examination showed that bladder HA-PLCL and PLCL were higher in the 4 weeks after operation than in the control group,while the bladder compliance of the HA-PLCL group was better.At post-operation 8 weeks,the bladder capacity of the animals in the 3 groups was higher than 4 weeks.Among them,HA-PLCL group had the largest average bladder capacity,which had the significant difference with blank group,but no significant difference between PLCL group(p<0.05).Conclusion:Hyaluronic acid can be used to produce HA-PLCL coaxial electrospinning material,HA-PLCL has a stable mechanical properties and degradation rate in vitro.The hydrophilicity of HA-PLCL is high,which is beneficial to the cell adhesion,growth and migration.The tissue toxicity of HA-PLCL is low,and it can be useful for keeping bladder capacity.