Research Of TGF-β1 Combined With IGF-1 Induced BMSCs Seeded On Composite Silk Fibroin/collagen Scaffold To Construct Functional Cartilage Tissue

Objectives:Articular cartilage injury is one of the serious diseases impact on human life,and also the age begin to become younger. Current clinical treatment methods include autologous or allogenic cartilage transplantation, but can cause new damage to the donor area and the source of donor is limited. This work is based on the previous studies, with collagen and silk protein as raw materials by freeze drying method to make scaffolds. Then the TGF-β1 and IGF-1 induced BMSCs as seed cells to construct tissue engineering on the scaffolds, in a microenvironment to induce chondrogenic differentiation of BMSCs.Methods:1.To separate BMSCs with density gradient centrifugation method and cultured in vitro and observe its characteristics.2.Cartilage direction induced and identification: To take third generation BMSCs and joined the chondrogenic induction liquid and toluidine blue and safranin O staining of red pigment was performed after 2 weeks later,.3.Preparation of silk fibroin/collagen scaffold for tissue engineering: To prepare silk protein and collagen with silkworm cocoon and beef tendon, blend the two solution with different proportion and used freeze-drying technique to make composite scaffolds(number1,2,3group).The porosity, water absorption expansion rate, mechanical properties, and pore size were detected. The third generation BMSCs were seeded onto the scaffolds, and were cultured for 14 days. The cell morphology and distribution were observed by MTT, HE staining and scanning electron microscopy(SEM).4.Tissue-engineered cultivation model construction: To construct tissue engineering model. The subjects were divided into three groups: group A(combined induction), group B(non induced) and group C(control group).5.To use alcian blue method for the determination of the supernatant of the culture medium acidic glycosaminoglycan in vitro cultivation model.6.To make immunohistochemical to detect cell-scaffold model expression level of type II collagen after 2 weeks culture.Results:1.BMSCs was successfully separated by density gradient centrifugal method and cultured three passages showed high purity without impurities.2.Toluidine blue staining and safranin O staining of red pigment wereperformed and showed the BMSCs have good multi-directional differentiation potential.3.The porosity of groups 1, 2 and 3 was respectively 94.6%±1.6%,80.6%±1.1% and 60.6%±1.0%, significant differences between three groups(P<0.05). The water absorption expansion rates of groups were respectively1623.7%±186.6%, 991.0%±151.6% and 559.6%±161.4%, showing significant difference among three groups(P<0.05). The elasticity modulus of groups were respectively(23.1±6.3)k Pa,(25.1±5.1)k Pa and(29.8±6.9)k Pa, showing no significant difference among three groups(P>0.05). The pore size of groups was respectively(189±12)μm,(110±15)μm and(60±16)μm, showing significant difference among three groups(P<0.05). MTT results showed that the cell proliferation in the group 1 was better than groups 2 and 3(P<0.05); HE staining showed the scaffolds staining cells in1 group were significantly more than groups 2and 3; even pore size, good intercommunicating of holes, and cells growth on the scaffolds in group1 under SEM, but small pore size, poor intercommunicating of holes and poor cell growth in group 2 and 3.4.GAG of culture supernatant of group A secernent was significantly higher than group B and C(P<0.05). GAG daily secretion of group Aincreased gradually,but group B and C always showed low expression tendency. A group confirmed chondrogenic differentiation of cells.5.A group expressed type II collagen, but group B and group C did not show type II collagen.Conclusions:1.BMSCs with multi-directional differentiation can be separated successfully.2. Silk and collagen have better biological compatibility and can obtain better pore connectivity, moderate pore size of the composite scaffolds, To explore the silk fibroin /collagen scaffold in two kinds of material quality ratio preliminary and a preliminary study on the properties of composite scaffolds and point out the direction for further research.3.TGF-β1combine with IGF-1 induced BMSCs were seeded onto scaffolds and can successfully construct vitro tissue-engineering cartilage morphology model.After two weeks of culture, A group model successfully show ability to form cartilage.