Study On The Functional Reparation Of Rat Uterine Horns Following Full-thickness Injury

Background:A variety of diseases or trauma can cause uterus severe damages which may lead to scar formation and subsequent amenorrhea, infertility or abnormal placenta implantation. There is lack of effective treatments to such severe damages of endometrium. Tissue engineering may offer a new method for uterine functional reparation. In the current studies, we used tissue engineering methods in the reparation of rat uterine horns with different healing stage (acute trauma or scar) to evaluate the effect of collagen scaffolds, growth factors and stem cells in the regeneration of uterine endometrium, muscular cells and blood vessels, remodeling of uterine scars and recovery of pregnant ability.Chapter One:The effect of collagen scaffolds loaded with basic fibroblast growth factor in the reparation of the new trauma of rat uterine horns following full-thickness injuryObjective:To evaluate the effect of collagen scaffolds loaded with PBS, NAT-bFGF or CBD-bFGF in the reparation of the new trauma of rat uterine horns after a1.5cm×0.5cm (about half of the length and circumference) full-thickness mechanical injury.Methods:Totally81female Sprague-Dawley rats were assigned to five groups, including sham operated group (uterine horns were intact without excision), spontaneous regeneration group (after uterine excision the defects were left open without reparation), collagen/PBS group (after uterine excision the defects were sutured with collagen membranes), collagen/NAT-bFGF group (after uterine excision the defects were sutured with collagen membranes loaded with NAT-bFGF) and collagen/CBD-bFGF group (after uterine excision the defects were sutured with collagen membranes loaded with CBD-bFGF).30and90days postoperatively, the animals were sacrificed and the uterine horns were prepared for the patency test and gross examination of adhesion, neovascularization and hydrometra. The uterine specimens were fixed with formalin. Sections of the damaged uterus were prepared for HE, Masson's trichrome and immunohistochemistry staining.90days postoperatively, animals were mated with male Sprague-Dawley rats to assess the fertilization ability.Results:(1) Patency test showed that spontaneous regeneration group had lower patency rate (45.00%) compared with Sham-operated group (100%), collagen/NAT-bFGF group (85.00%) and collagen/CBD-bFGF group (95.00%).30days after the surgery, the appearance of collagen/CBD-bFGF group was closer to adjacent tissue and had apparent neovascularization compared with collagen/PBS group.90days after surgery, all remodeled sites resembled normal-appearing tissue on inspection and in texture.(2)30days and90days after surgery, the thickness of endometrial in collagen/CBD-bFGF group was similar to sham operated group and was higher than the other three groups.(3) Masson's trichrome staining showed that collagen/CBD-bFGF group had more collagen remodeling, more apparent neovascularization and cellularization and less collagen deposition than that of collagen/PBS group. Endometrial of spontaneous regeneration group had abundant collagen deposition and scar formation.(4) Immunohistochemistry staining showed that the percentage of a-SMA positive area, blood vessel density and the number of proliferative cells in collagen/CBD-bFGF group were significantly higher than that in spontaneous regeneration group, collagen/PBS group and collagen/NAT-bFGF group.(5) Pregnancy test showed that the pregnancy rate of collagen/CBD-bFGF group was close to that of sham operated group (100%) and much higher (86.67%) than those of spontaneous regeneration group (33.33%) and collagen/PBS group (33.33%). There was no statistical difference between the collagen/CBD-bFGF group and collagen/NAT-bFGF group (60.00%). Furthermore, in collagen/CBD-bFGF group embryos were found both in grafted and normal tissue in2uterine horns (2/15). But in the spontaneous regeneration group, collagen/PBS group and collagen/NAT-bFGF group, embryos were present in normal uterine tissue instead of within the grafted tissue.Conclusion:The collagen loaded with CBD-bFGF could notably improved regeneration abilities of uterine endometrium, blood vessels and smooth muscle cells as well as better pregnancy outcomes.Chapter Two:Study on the effect of vascular endothelial growth factor on the remodeling of scarred rat uterusObjective:To evaluate the effect of CBD-VEGF on the remodeling of scarred rat uterus following full-thickness injury.Methods:After1.0cm×0.5cm excision of the uterine horns of72female Sprague-Dawley rats, the defects were left open for spontaneous healing.30days after the operation, uterine scar formation was confirmed by a second incision. The rats with scarred uterus were assigned to three groups, including PBS group (50μL PBS was injected into the surrounding tissue of uterine scar), NAT-VEGF group (0.25nmol NAT-VEGF was injected into the surrounding tissue of uterine scar) and CBD-VEGF group (0.25nmol CBD-VEGF was injected into the surrounding tissue of uterine scar). At3hours and6hours after injection, exogenous VEGF in the scared area was detected by western blot using anti-polyhistidine.30and60days after the injection, the animals were sacrificed and the uterine horns were prepared for the gross examination of adhesion, neovascularization and intratubal obstruction. Uterine specimens were fixed with formalin. Sections of the scared uterus were stained for HE and immunohistochemistry staining.60days after the injection, animals were mated with male Sprague-Dawley rats to test fertilization ability. Animals were euthanasia at mid-to late gestation, and uterine horns were examined for the position of embryos.Results:(1) At30days after injury, the uterine horns formed scars.(2) At3hours and6hours after injection of PBS, NAT-VEGF and CBD-VEGF, Western blot showed that there was no the exogenous VEGF in the PBS group and the level of exogenous VEGF in the CBD-VEGF group was significantly higher than that in the NAT-VEGF group.(3)30and60days after injection, the deposition of collagen in the CBD-VEGF group was reduced, and the thickness of endometrial and the number of glands in CBD-VEGF group was higher than that in PBS and NAT-VEGF groups.(4) Immunohistochemistry staining showed that the percentage of a-SMA positive area and blood vessel density in CBD-VEGF group was higher than that in PBS and NAT-VEGF groups. Muscle bundles in CBD-VEGF group were more organized than other groups.(5) Pregnancy test showed that the pregnancy rate in the scared area of CBD-VEGF group (50.00%) was much higher than those of PBS (6.25%) and NAT-VEGF groups (18.75%).Conclusion:Injection of CBD-VEGF into uterine scar tissue resulted in a high local concentration and prolonged biological effect of the growth factor. CBD-VEGF notably promoted vascularization and remodeling of the scarred uterus and improved uterine fertilization ability.