Controlled Release Of IGF1 From Microbeads Enhances Urethral Sphincter Function And Histological Structure In The Treatment Of Female Stress Urinary Incontinence In A Rodent Model

BackgroundStress urinary incontinencecan be ameliorated if the injured external urethral sphincter(EUS)can be restored.Insulin-like growth factor-I(IGF-1)acts as a central regulator of muscle regeneration and recovery.Construction of the vector,which can stabilize and control the release of IGF-1 to maintain the efficiency of slowing the release of active drugs,can promote the recovery of damaged tissue and help patients with stress urinary incontinence to restore the function of urinary control more effectively.A novel controlled drug release system,alginate-poly-L-ornithine-gelatin(A-PLO-G)microbeads,to serve the purpose of IGF-1 delivery to enhance the recovery of injured tissue.Purpose1 To fabricate the A-PLO-G microbeads and examine their physiochemical property;release study of IGF-1 from microbeads was performed;2 To eject the IGF-1 microbeads to the VD rats and use LPP,EUS EMG and histology test to explore the therapeutic potential of IGF-1 loaded A-PLO-G multiple layers microbeads.Mehtods1 Sustained-release microspheres could be prepared by emulsion crosslink technique.Sodium alginate solution would be dispersed as small droplets alone through making a template and then become the calcium alginate microbeads after coupling with calcium cation in calcium chloride solution.Calcium alginate microspheres coated IGF-1 in turn would be mixed with polyornithine and gelatin in turn to make the IGF-1 loaded A-PLO-G microbeads.The diameter of the microbeads morphology and particle analysis were observed under light microscope.The in vitro stability at room temperature,external mechanical strength and biological safety analysis needs to be detected.And ELISA kit was used to calculate encapsulation efficiency,drug loading and release characteristics of IGF-1 loaded microbeads.2 Forty-four nulliparous female adult Sprague-Dawley rats(10-13 weeks,240-290 g)were randomized into 4 groups.Thirty-three rats underwent VD and were treated with either IGF1 loaded A-PLO-G microbeads(VD+IGF1 microbeads),empty A-PLO-G microbeads(VD+empty microbeads),or normal saline(VD+saline).Eleven rats in the sham injured group underwent sham VD and were treated with saline(Sham).After VD,IGF-1 microbeads and empty microbeads were injected immediately while the rats were still under anesthesia.Functional outcomes were assessed 1 week later and included leak point pressure(LPP)and external urethral sphincter(EUS)electromyography(EMG)from 1 second segments of continuously recorded data at baseline and at the peak pressure during LPP testing.Animals were euthanized immediately after functional testing,and then the urethra and anterior vagina will be dissected en bloc for anatomical analysis via histology and immunohistology.Results1 The diameter of 90%of the microbeads was from 90 to 100?m and the average diameter was(91.72±3.51)?m;The diameter of the microbeads increased a little after being incubated in vitro for 4-12 days.The average diameters of microbeads were(92.34±3.73)?m,(95.62±4.43)?m,(96.91 ±4.76)?m in 1,4,12 days respectively.The microbeads were undissolved and stable in saline solution.After shaking the beakers with microbeads in saline solution at a speed of 100RPM for 6,12,24,36,48,60hours respectively,the integrity rate of microbeads were 100%?100%?100%?99.7±0.2%?99.2±0.8%?98.4±0.3,which showed good stability and implied that microbeads can resist a greater degree of extrusion and shaking after transplanting into patients' bodies;The OD of the cells cultured in microbeads leaching solution with different concentrations(100%?50%?20%)were 0.561±0.0576?0.578±0.031?0.581 ±0.012 respectively,which has significant difference compared to positive control group(p<0.05).The cytotoxicity grades were 0 to 1,calculated through the relative growth rate of RGR,which proved that microbeads had no cell toxicity;calculating the release concentration of IGF-1 through OD with ELISA kit shows that microbeads released IGF-1 faster in the first two days and had a characteristic of "burst".A 40%of IGF-1 released from the microbeads and about 90%of IGF-1 cytokines were released into the solution after 36 hours.The 14 days release curve showed a linear increase trend;The loading of microbeads is 606.52ng/g.The entrapment efficiency was 45.3%.All of them were in accordance with the requirements of the design of microbeads.2Leak point pressure was significantly increased after VD in rats treated with IGF1A-PLO-Gmicrobeads(28.4±1.2 cmH2O),compared to those in saline and empty microbeads(23.9±1.3 cmH2O,21.7±0.8).Histological analysis demonstrated well-developed,well-organized skeletal muscle fibers in external urethral sphincter in the rats treated with IGF1A-PLO-Gmicrobeads,which was similar to that of sham-injured animals.In contrast,substantial muscle fiber attenuation and disorganization was observed in VD injured,NS-or empty microbeads-treated rats.ConclusionIn summary,this study successfully prepared reliable A-PLO-G microbeads and their preparation conditions were optimized.The A-PLO-G microbeads were stable,mechanically strong,and biological safe,which means they could meet the need of transporter for IGF-1.The microbeads' entrapment efficiency and release rate of IGF-1 also in accordance with the requirements of the design of microbeads.Using the VD as the SUI model,this study demonstrated that controlled releases and local administration of IGF1 fromIGF1-A-PLO-G-beads provide a possible approach to facilitating recovery from SUI induced by simulated childbirth injury,likely mediated via preservation and regeneration of the vasculature.IGF1 A-PLO-G-beads thus represent an attractive,alternate approach in the treatment of female SUI.In future projects,we will compare this immediate treatment to delayed treatment that mimics current cell-therapy in the clinical situation.