The Cytocompatibility Study Of Silk Fibroin/Poly-l-iactic Acid Scaffold And Chondrocytes In Vitro

Cartilage is a part of the human body normal tissue, which constitutes the mainframe structure in early embryogenesis。Generally，it is be replaced by bone in grownman.For adulthood, cartilage is only scattered distribution.Cartilage is an importantpart of the joints,which plays an important role in friction buffer、reducing vibrationand keep the normal joint movement.However,chronic changes,such as injury,inflammation, often make articular cartilage damage, thus cause changes in theanatomical structure of the articular cartilage, which affect the joint movement,resulting in a series of joint symptoms, such as joint pain, joint swelling, jointhemorrhage, etc.The temporomandibular joint (TMJ) is closely related to the base of the joint(dynamic) joints, with multiple axis of motion, swivel and slide, participate in avariety of physiological functions such as mastication, swallowing, speech andexpression. When disorder,inflammation, injury or tumor invasion occurred in thearticular cartilage, the movement of temporomandibular joint (TMJ) is affected, forthe defecting condylar cartilage can not self-healing.Tissue Engineering was first held in Washington in1987in the biologicalEngineering team meeting of United States science foundation,which is integratedapplication of the basic principle of the basic theory, basic technology and basicmethod of Engineering and life science, pre-builting a biological active implant in vitro, then implanted, replacing part or all of the Tissue and organ function. There arethree core elements: tissue engineering scaffold material, seed cells and growthfactors.Silk fibroin (SF) is a widely studied renewable natural organic polymer scaffoldmaterials. Polylactic Acid (PLA) is approved by the food and drugadministration(FDA) as a kind of biological materials.Both have the good tissue andcell compatibility, non-toxic harmless, renewable use,etc.Previous animal acutetoxicity experiment and in vitro cytotoxicity test, confirmed that silkfibroin/poly-L-lactic acid(SF/PLLA) made of method of electrostatic spinning hasgood biocompatibility.Based on previous research results, we co-culture the rabbit knee jointchondrocyte with silk fibroin/poly-L-lactic acid(SF/PLLA) scaffold，hematoxylin-eosin staining and Alcian blue-nuclear fast red staining are tested on day3、7、14and21respectively. Cell adhesion is tested by Scanning electron microscopy(SEM). Cell proliferation was Determined by MTT test.Specific content is as follows:1、Co-culture cartilage cells and scaffold materials.Co-culture the cartilage cellsof second generation of logarithmic phase with silk fibroin/poly-L-lacticacid(SF/PLLA) scaffold materials,on the early stage of co-culturing, the cartilage cellsare uniform distribute which can be circle visible on silk fibroin/poly-L-lacticacid(SF/PLLA), part round cartilages disappeared after4h, cartilages invisible exceptfor the edge of scaffold materials after24h. 2、HE staining results of cartilage cells-scaffold materials co-culture.On3day,cells are relatively loose distributing, but adhesion well, intercellular substancestaining is shallow; on7days, the cell number increased, nuclear division is visible;on14days, cartilage lacuna" is visible,the staining of intercellular substance isobviously deepened; on21days, the staining of cartilage cells and intercellularsubstance are interconnection.3、Alcian blue-nuclear fast red staining results of co-culturing cartilagecells-scaffold materials.On3day,cartilage cells are small amount, intercellularsubstance(mucopolysaccharide) staining is shallow；on7day, cartilage cells increased;on14days, carticlage lacuna" and cartilage capsuleis are visible, and staining isdeepened, but not uniform; on21day, the staining of cartilage cells and intercellularsubstance are interconnection.4、Scanning electron microscope results of co-culturing the cartilage cells-scaffold materials. On3day, cells are scattered, some cells are deep inside the aperture, the surface of cell membrane is smooth, and cell matrix is less; on7day, the cells have nest-like pseudopodia and cellular matrix much more distribution.Cellular matrix is filling in the bracket aperture.5、MTT test. We set SF/PLLA+cartilage cells group as the experimental group,PLLA+cartilage cells group as control group, chondrocytes cells only as blank group,each group set six complex hole, measuring absorbance values of490nmwavelength.Get rid of the highest in each group respectively with the lowest as thefinal absorbance value in each group. Use SPSS19.0statistical software analysis. The experimental group get more absorbance value, which is more conducive to celladhesion and proliferation.The results show that the cartilage cells can obtain good adhesion on silkfibroin/poly-L-lactic acid(SF/PLLA) scaffold materials; the quantity of cartilage cellsand the cell matrix are increased as time goes on, but the cartilage cell phenotype isnot changed;MTT data showed the cartilage cells on silk fibroin/poly-L-lacticacid(SF/PLLA) scaffold materials can obtain better adhesion and proliferation.