Study On Biocompatibility And Mechanical Property Of Silk-PLGA Scaffold

Anterior cruciate ligament (ACL) is an important structure for the knee stability. Poor healing capacity of the ACL after injury has led to necessity of reconstruction with various grafts under arthroscope in most of cases. However, those graft materials own varied disadvantages. Tissue engineering may potentially provide solution for these problems. The ideal scaffolds for tissue engineering should have good biological safety and organization of compatibility, can provide a reliability strength for ligament in early time, and provide space for growth, reproduction, and physiological function of seed cells play. Through these experiments of cell toxicity, biological safety, mechanical strength, biocompatibility, and scaffold degradation liquid with cell proliferation, silk -PLGA scaffold is an ideal scaffold material for tissue engineering.Part oneObjective:To evaluate the cytotoxicity and biological safety of Silk-PLGA braided scaffold materials for providing the scientific and experimental basis for clinical application.Methods:Silkworm silk fibroin fiber was mixed with PLGA filament at ratio of 2:3, then silk-PLGA scaffold was prepared by braiding them. Cell observation method and MTT assay were used to detect the cytotoxicity of silk-PLGA braided scaffold with extraction solutions. The cytotoxicity of the scaffold extract at concentrations of 25%,50%,and 100% to bone marrow-derived mesenchymal stem cells (MSCs) was detect by MTT assay. The bio-safety of the silk-PLGA braided scaffold materials were assessed according to the relevant standards by acute systematic toxicity test, intracutaneous irritation test, hemolysis test, and pyrogen test. The experimental results were analyzed and assessed by criterion.Results:MSCs grew well in the experimental groups and negative control group and there was no significant difference in cell morphology between these groups. The relative growth rates of the experimental groups were all over 93%,and the cytotoxicity grade was at level 0 or 1 at day 1,3,5,and 7 after cultivation,according to the standard of cytotoxicity.So the silk-PLGA braided scaffold is basically non-toxic. In acute systematic toxicity test of the silk-PLGA scaffold materials, no obvious toxic reaction was observed. In intracutaneous irritation test, no intracutaneous irritation reaction was found. In hemolysis test,the hemolysis rate of the silk-PLGA scaffold materials was 1.17% and was less than 5%.It showed that the scaffold materials was no hemolysis. In pyrogen test,the highest temperature increment of experimental rabbit were 0.4,0.2,0.3℃,respectively,so there was no pyrogen reaction.Conclusion:Silk-PLGA mixed scaffold materials have good biological safety and cytocompatibility. The of the scaffold material satisfies the safety standard. It is a potential scaffold material for tissue engineered ligament or tendon.Part twoObjective:To evaluate mechanical property of scaffold braided by Silk-PLGA and its cytocompatibility with rabbit bone marrow-derived mesenchymal stem cells(MSCs), and investigate the feasibility of fabricating tissue-engineered ligament/tendon by using such scaffold.Methods:Firstly, a hybrid rope-shaped and mesh-shaped silk-PLGA scaffold containing monofilament 6144 was prepared by twisting weave using a mixture of silkworm silk fibroin fiber and PLGA filament with a ratio of 2:3, and silk fiber-PLGA fiber filaments were then modified by fibronectin. The biomechanical property of the above-mentioned silk-PLGA scaffold was measured with the electric universal testing machine. Secondly, rabbit MSCs were isolated and proliferated in vitro. Then, they were seeded on the surface of the scaffold. The situation of cell growth, matrix formation, as well as the combination of cells and scaffold was observed under an inverted phase contrast microscope and scanning electron microscopy (SEM).Results:The appearance of the scaffold braided by silk-PLGA is milky white, spiral rope-like, and homogeneous with a strong obdurability. The diameter of the scaffold is 2.3 mm. The maximum load, tensile strength, breakpoints elongation rates, and elastic modulus of the scaffold were (315.06 ± 30.77)N,(75.83 ± 7.46)MPa,(61.39±7.26)%,(213.58± 23.45)MPa,respectively. Scanning electron microscopy observation revealed that rabbit MSCs grew and adhered well. Most of the MSCs showed spindle-shaped with good stretch, and they grew in three dimensions. Moreover, the MSCs secreted rich cell matrix on the surface of the scaffold braided by silk-PLGA. The appearance of the scaffold knitted by silk-PLGA is milky white, mesh-like, and homogeneous with a strong obdurability. The width of the scaffold is about 5 mm. The maximum load, tensile strength, breakpoints elongation rates, and elastic modulus of the scaffold were (118.32±11.21)N,(52.90±5.03)MPa,49.79%±5.16%,and(177.25±19.18)MPa, respectively. Scanning electron microscopy observation revealed that rabbit MSCs grew and adhered well. Most of the MSCs showed spindle-shaped with good stretch, and they grew in three dimensions. Moreover, the MSCs secreted rich cell matrix on the surface of the scaffold knitted by silk-PLGA.Conclusion:The scaffold braided and knitted by silk-PLGA has good biomechanical properties and cytocompatibility. Therefore, it is of great potential for tissue engineered ligament and tendon.Part threeObjective:To observe the effects of degradation fluid of silk-poly(lactic-co-glycolic acid) braided scaffold on the proliferation and growth of rabbit bone marrow-derived mesenchymal stem cells in vitro.Methods:The silk-poly(lactic-co-glycolic acid) braided scaffold was degraded in complete culture medium for 14 weeks in vitro, and medium was replenished every week. The pH value of degradation fluid of the scaffold was determined every week. Rabbit bone marrow mesenchymal stem cells were cultured with degradation fluid and complete medium each 100μL (experimental group), while negative control group was added with complete medium 200 μL, culturing for 4 days.Results and Conclusion:(1) pH value changes of degradation fluid of silk-poly(lactic-co-glycolic acid) scaffold:pH value declined slowly with the first 3 weeks, from 7.00 to 6.89. However, the value went down quickly at 4 weeks, maintained a low level at 6-11 weeks, between 5.16 and 5.67. Then pH values increased at 12-14 weeks, rising to 6.95. (2) Morphology of mesenchymal stem cells:The proliferation and growth of mesenchymal stem cells in experimental group and negative control group were basically similar. Degradation fluid inhibited the cell growth at degradation 7-10 weeks, exhibiting relatively less counts and sparse arrangement of the cells, while degradation fluid in other weeks had no obviously inhibition on the cell growth. (3) Proliferation of mesenchymal stem cells:Degradation fluid of the scaffolds at 1-6 weeks and 11-14 weeks had no significant impact on cell proliferation, and the relative growth rates of the cells were all over 92.1%, with toxicity grade 0 or 1 level; degradation fluid at 7-10 weeks had an inhibition effect on cell proliferation, but relative growth rates were 82.5%-87.9%, with toxicity grade 1 level, as qualified. Experimental findings indicate that, the degradation fluid of silk-poly(lactic-co-glycolic acid) braided scaffold has good cytocompatibility.