| Part I Preparation and Evaluation properties of degradable biocompatible polymers scaffolds composed of polylactic acid-co-glycolic acidObjective:Fabricating three-dimensional degradable scaffolds with biocompatible polymers composed of polylactic acid-co-glycolic acid (PLGA) using the electrospinning methods. Take photos and analysis the structure of the samples with scanning electron microscopy. The tensile and suture strength were tested in vitro respectively, hydrolytic degradation was carried out in phosphate buffer saline of pH 7.4 at 37?. Cell cytotoxicity was evaluated by MTT measurement and the proliferation of cells was evaluated by BrdU Eliasa measurement. Building subcutaneous embedding models and study the histocompatility of the PLGA scaffold. Explore its feasibility as a cardiac tissue engineering scaffolds.Methods:1. Preparation of PLGA scaffolds and ultrastructural observation: PLGA electrospun was using PLGA (50/50) particles, with chloroform/ DMF (volume ratio 2:1) as solvent mixture, dubbed the concentration of 8% solution, the solution was stirred at room temperature overnight. When the polymer was spinning, the solution was inserted into 30ml glass syringe with 8# needles, spinning voltage of +10 kV, receiving distance was 12cm, feed rate of 3ml/h, receiver was the copper covered with foil, and produced the electrospun scaffolds. Spinning the samples obtained after vacuum spraying by SEM and analysis the surface morphology, accelerating voltage of 20 kV. 2. The suture and tensile strength of PLGA films was tested. PLGA films were trimmed into the 20mm×5mm rectangle (n=7), then one end of the clip in the fixture of the tensile machine, the other end with the 4# surgery line was stitched with a piece of cloth to test their strength suture. The PLGA films was cut into I-shaped spline (n=8), caught in the tension machine at both ends of the fixture, the test range of length 20mm, width 2mm, thickness of 0.5mm, and test the breaking strength.3. The hydrolytic degradation test of PLGA films was carried out in vitro. The PLGA films were cut into square samples (n=3×4) with size of 10mm×10mm, decontamination, distilled water washed, dried and weighed, then placed into 6 well plate, added PBS buffer, complete infiltration of samples, placed in incubator at 37?. Respectively after 1, 2,4,6 weeks, the samples were removed, washed with deionized water to dislodge the salt ions of their surface, and then vacuum drying at room temperature to constant weight, and calculated the weight loss.4. PLGA films cell compatibility testing:Organic tin stabilizers of polyvinyl chloride (PVC) for the positive control group, the collected PVC, PLGA films were trimmed to 10mm×10mm size for washing, disinfection, in accordance with the membrane surface area:extraction transmitter=3 cm2:1 ml in proportion to prepare the extract. L929 cells culture in extracts using MTT cytotoxicity test method, using BrdU cells proliferation method, cells morphology was observed with the direct contact culture method to evaluate the cells compatibility.5. PLGA films histocompatibility testing:The PLGA films were cut into samples of about 10mm×10mm size (n=3×3), establish rat subcutaneous models, respectively after 1,2,4 weeks, remove the samples, fabricate the tissue sections and HE staining, observe the material changes. Results:1. Silk spuns of PLGA film have good shape, spin size is homogeneous and internal pore interconnected. The pore size ranging from about 100 to 150 microns, and pores were evenly distributed, favor for the exchange of nutrients and the excretion of metabolic products, prone to meet the demand for cells growth.2. Mechanical test results in vitro of PLGA electrospun films were as follows:suture strength was (4.9±0.6) N, breaking tensile strength was (1.6±0.3) MPa, and meet the mechanical requirements of cardiac patch.3. The weight loss of PLGA films is found in phosphate buffer, mass loss was 13.4% after 6 weeks, and the degradation of scaffolds accelerated from the 4th week. In addition, we also observed that the sacffolds appear collapse solution after 8 weeks, indicating that the PLGA scaffold with biodegradable.4. Cytotoxicity test demonstrate that the toxicity of PLGA stabilized at in the 0?1 level, the positive PVC control group stabilized at 4 levels, indicating that the PLGA materials is less toxic and conducive to cells growth. L929 cells experienced a period of rapid DNA synthesis from 2 to 5 days from the BrdU Elisa results and actived proliferation capacity. Morphological observation showed that L929 cells were spindle-shaped or triangular stretch in the PLGA membrane, adhered well, vigorous growth, suggesting a good cellular compatibility of the PLGA material, consistent with the requirements of myocardial tissue engineering.5. Embedded biopsy specimens of subcutaneous observed it consistent with chronic inflammation. And rat fibroblast cells migrated into the internal of the PLGA scaffold, proliferated, arranged along the spinning fibers and formed a dense structure after 4 weeks, and that demonstrated the PLGA scaffold has good histocompatibility. Conclusions:1. The PLGA electrospun scaffold has a good micromorphology to meet the geometric requirements for cardiac tissue engineering.2. The PLGA electrospun scaffold with good seam strength and tensile strength, meet the mechanical requirements for myocardial tissue engineering scaffolds.3. The PLGA electrospun scaffold is biodegradable, the degradation accelerates along with time.4. The PLGA scaffold had no significant cytotoxicity, seed cells proliferated well in the extract, and the morphology also confirmed the good cell compatibility of the scaffolds.5. PLGA electrospun scaffold with good biocompatibility, appeared no significant inflammation and rejection after implantation, and can be safely transplanted in vivo. Part II The effect of the implantation of cardiac patches combined with auto-omental transplantation in MI modelObjective:Ischemic heart disease with high morbidity and mortality, remains a major threat to human health in our country, and myocardial cells appear apoptosis after myocardial infarction, leading to heart failure. Although many treatment methods, such as medical therapy, percutaneous transcoronary angioplasty and CABG surgery, can improve heart function, but there are insufficient. Cells transplantation is a promising choice for the treatment of ischemic heart disease. There are a large number of experimental reports, and the most current treatments utilize injection way, but there transplantation efficiency is still low, and need to find new effective treatment means. The tissue engineering medicine provides us with a new idea:fabricating new biodegradable PLGA three-dimensional scaffolds in vitro, seeding bone marrow mesenchymal stem cells in the scaffolds and constructing composite tissue engineered graft to improve the efficiency of cells transplantation for MI treatment. For the defect of blood supply after transplantation, we used pedicled omental wrapping operation (OP) to improve the local micro-environment and provide blood supply for the grafts. We evaluate the efficacy of the treatment measure in rat MI model and provide experimental basis for the future clinical application.Methods:Male Sprague-Dawley (SD) rat bone marrow MSCs were separated though there differential adhesion property on plastic surface. Culture, select and label the 3rd generation of vigorous growth MSCs with 4',6- diamidino-2-phenylindole hydrochloride (DAPI). Labeled cells were seeded in the PLGA scaffolds and cultured for 4 days in vitro. Female SD rats 4 weeks old were selected, selected the left anterior descending coronary artery ligation to establish rat MI models. One week later, transthoracic echocardiography screening, the qualified female MI rats with left ventricular ejection fraction (LVEF) less than 60% and left ventricular shortening fraction (LVFS) less than 30% were randomly divided into 4 groups:the control MI group (n=10), MSCs-PLGA group (n=12), PLGA-OP group (n=12) and MSCs-PLGA-OP group (n=35), for the second operation. MI control group underwent secondary thoracotomy; MSCs-PLGA group underwent epicardial tissue engineering patch graft on infracted myocardium; PLGA-OP group underwent epicardial cell-free PLGA patch composite with OP; MSCs-PLGA-OP group were transplanted composite engineering grafts with OP. Four weeks after transplantation operation, echocardiography was used to evaluate the left ventricular function, and then the hearts were harvested for pathological examination, such as H&E staining, immunohistochemistry and immunofluorescence staining. In addition, the MSCs-PLGA-OP group rats were killed for the cytology observation separately after 1,2 weeks of the second operation.Results:After the operation, the rats survived in good condition, and there are no significant side effects observed. Echocardiography showed that compared with the MI control group (49.85%±5.03% & 22.06%±2.78%), both the LVEF and LVFS of MSCs-PLGA-OP group (59.68%±5.78% & 27.65%±3.81%) and MSCs-PLGA group (56.89%±7.57% & 26.22±4.55%) were improved (p<0.05, ANOVA test). Although the PLGA-OP group (55.46%±3.75% & 25.28%±2.22%) has a trend of heart functional improvement, but there is no statistical significance compared with the control group. Both LVEDD and LVESD of all groups were no significant difference, indicating that PLGA spinning flexible scaffolds do not affect the cardiac systolic and diastolic function. Observation from 1, 2,4 weeks after the second operation, the transplanted cells apoptosis with time, but stabilized from 2 to 4 weeks in MSCs-PLGA-OP group, indicating that the transplanted omentum has provided sufficient blood supply for the cells, and immunohistochemistry also confirmed this point. There are also many MSCs observed migrated into the infarct area after 4 weeks of transplantation operation, and a small amount of cells differentiated into cardiomyocytes.Conclusion:Hybrid three-dimensional tissue engineered scaffolds seeded cells with the pedicled omental wrapping operation can improve cells transplant efficiency, improve local micro-environment in infarct area, and improve heart function for the MI treatment. |
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