Experimental Study Of Urethral Tissue Regeneration Induced By A New Functional Biomaterial

Background Urethral defects is a class of common urinary diseases. It is one of the effective therapies for this kind of diseases to reconstruct the defect surgically. Reconstruction of complex urethral defect is one of the most challenging techniques for urologist. Choosing suitable materials is the key factor to influence the outcome of urethral reconstruction. Autologous tissues are most common source of urethral alternative materials clinically, but they can’t meet the requirement of clinic at all the time, the complications of the sugery can’t be controlled effectively too. Constucting suitable functional biomaterial is promised to solve these problems which can’t be solved by autologous tissues, and becomes reliable source of urethral substitutes. Urethra is rich in blood vessels, rich blood supply is essential to keeping normal morphology and function of urethra, and promoting urethral wound repair and urethral tissue regeneration. VEGF is a specific mitogen for vascular endothelial cell, and has showed potential application to promote urethral tissue regeneration. In the study of constucting functional biomaterials with VEGF, how to combine the factor with material effectively, and establish a target binding-release system is a key issue. Building a persistent and efficient micro environment of VEGF will help to promote tissue regeneration. Collagen binding domain(CBD) can bing to collagen specifically. Conjugating CBD to VEGF via genetic engineering methods, could make VEGF get the ability to bing to collagen specifically. On this basis, utilizing collagen as matrix, CBD-VEGF as bioactive factor, we can constructe a new functional biomaterial which can provide three-dimensional structure and continual stimulating signal of VEGF for tissue regeneration. When it was used to reconstruct urethral defect, the collagen biomaterial modified with CBD-VEGF would promote urethral tissue regeneration more effectively, and improve the outcome of urethral reconstruction.Materials and methods 1.The expression, purification and identification of recombinant protein native vascular endothelial growth factor(NAT-VEGF) and CBD-VEGF. Via vector p ET28 a and Escherichia coli BL21(DE3), we built an effective prokaryotic expression system for NAT-VEGF and CBD-VEGF. The recombinant plasmids of p ET28a-VEGF and p ET28a-CBD-VEGF were transformed into the Escherichia coli BL21(DE3), and the recombinant protein of NAT-VEGF and CBD-VEGF were induced to expression via IPTG. Then the recombinant proteins were purified through nickel column chromatography. Finally the the recombinant protein NAT-VEGF and CBD-VEGF were identified by SDS-PAGE electrophoresis. 2.Binding activity of recombinant protein NAT-VEGF and CBD-VEGF to collagen. The binding activity of recombinant protein NAT-VEGF and CBD-VEGF to collagen was evaluated by ELISA. Different concentration of recombinant protein(0, 0.094μM, 0.1875μM, 0.375μM, 0.75μM, 1.5μM, 3μM, 6μM) was dripted into enzyme labeled board coated with isopyknic type I collagen. Two hours after, the recombinant protein which didn’t bind to collagen washed with PBS. The recombinant protein was labelled by anti-VEGF antibody and anti-Rabbit Ig G. After staining, the absorbance at 405 nm was counted by microplate spectrophotometer. The result was used to evaluated the binding activity of the two recombinant proteins. 3.Releasing characteristic of recombinant protein NAT-VEGF and CBD-VEGF from collagen. The same amount of recombinant protein NAT-VEGF and CBD-VEGF was dripted into enzyme labeled board coated with isopyknic type I collagen respectively. Two hours after, PBS(500μl per well) was added into the enzyme labeled board. Then 400μl PBS was removed from each well, and 400μl fresh PBS was added into each well again every 12 hours. When accomplish binding and each 24 hours since then, the absorbance at 405 nm was counted by microplate spectrophotometer. The result was used to evaluated the releasing characteristic of recombinant protein NAT-VEGF and CBD-VEGF from collagen. 4.Biological activity of recombinant protein NAT-VEGF and CBD-VEGF. Human umbilical vein endothelial cells were cultured in 48-well culture plate uncoated or coated with isopyknic type I collagen. After culture for 8 hours, NAT-VEGF, CBD-VEGF and commercial VEGF(rh VEGF) were added into culture plate with the final concentration of 0, 0.094μM, 0.1875μM, 0.375μM, 0.75μM, 1.5μM, 3μM, 6μM. After another 72 hours of culture, proliferation of umbilical vein endothelial cells was tested by MTT assay. The result was used to evaluated the biological activity of recombinant protein NAT-VEGF and CBD-VEGF. 5.The role collagen biomaterials modified with CBD-VEGF in urethral tissue regeneration in a beagle’s extensive urethral defect model. Fifteen adult male beagles were randomly assigned to three groups, Collagen group, Collagen-CBD-VEGF group, MSCs group, 5 animals in each group. Five centimeters segment of the entire anterior urethra between bulbous urethra and baculum was cut off to establish the extensive urethral defect model. Then the urethral defect was reconstructed with simple collagen scaffold, collagen scaffold modified with CBD-VEGF, and collagen scaffold seeded with MSCs respectively. Urination after urethral reconstruction was observed for 6 months, then the morphology and function of the neo-urethra was evaluated with retrograde urethrography and urodynamic studies. Finally the neo-urethras were harvested, the tissue regeneration and matrix metabolism were evaluated by HE staining, masson-trichrome staining and immunofluorescence staining.Results 1.Recombinant protein NAT-VEGF and CBD-VEGF could be expressed utilizing gene engineering technique. The molecular weight of NAT-VEGF was about 18 k Da, the molecular weight of CBD-VEGF was about 20 k Da. 2.The recombinant proteins could be purified through nickel column chromatography. The purified CBD-VEGF could bing to collagen specifically, and steadily release from collagen. 3.CBD-VEGF could promote umbilical vein endothelial cells proliferation remarkably, its biological activity was similar to NAT-VEGF and rh VEGF. When binding to collagen, the biological activity CBD-VEGF was even higher than NAT-VEGF and rh VEGF. 4.CBD-VEGF and MSCs could all promote regeneration of epithelial tissue, the morphology of urethral epithelial layer of neo-urethra in the two groups was similar to normal urethra. CBD-VEGF and MSCs could all promote regeneration of smooth muscle too, but the amount of smooth muscle couldn’t reach the level of normal urethra, the distribution of the new smooth muscle was disordered. CBD-VEGF could promote regeneration of blood vessel tissue more effectively, the morphology of some neo-vessels was similar to sinusoid in normal corpus spongiosum. CBD-VEGF could reduce collagen deposition extracellular matrix significantly, and suppress contracture of neo-urethra. 5.CBD-VEGF and MSCs could all improve the outcome of urethral reconstruction. The mean diameter of the neo-urethra in CBD-VEGF group has no difference with that in MSCs group, the pressure of the neo-urethra in CBD-VEGF group was lower than that in MSCs group statistically.Conclusion Recombinant protein NAT-VEGF and CBD-VEGF could be expressed utilizing vector p ET28 a and Escherichia coli BL21(DE3). The biological activity of CBD-VEGF was similar to NAT-VEGF and rh VEGF. When binding to collagen, the biological activity CBD-VEGF was even higher than NAT-VEGF and rh VEGF. The purified CBD-VEGF could bing to collagen specifically, steadily release from collagen, and could establish a target binding-release system for VEGF. In the beagle’s extensive urethral defect model, CBD-VEGF could urethral tissue regeneration significantly, improve the outcome of urethral reconstruction. The effect of CBD-VEGF in urethral reconstruction has achieved and surpassed partly the effect of MSCs. Collagen-CBD-VEGF is a new choice to construct urethral substitutes for urethral reconstruction.