Fabrication Of PLLA-based Scaffold And Interaction Between Nanotopography Of Scaffold And Cells

In the present study, poly-L-lactide (PLLA) substrate scaffolds with differentnanotopographies were prepared by supercritical CO2phase separation technique,using ammonium bicarbonate (AB) particles as the porogen, and the interactionsbetween different nanotopographies and cell functions were also investigated.Firstly, scaffolds (14mm diameter,3mm thick) with different nanotopographieswere prepared under different conditions: PLLA was dissolved separately indichloromethane, dioxane and mixed dichloromethane:dioxane1:1(v:v), PLLAconcentration was10%(w/v), the porogen amount was AB/PLLA=20:1(w:w). Thescanning electron microscopy study demonstrated that the scaffolds presenteddifferent nanotopographies of thin wall structures with large pores, uniform fibrousstructures and mesh structures with both large and small pores. The PLLA porousscaffolds possessed a high porosity(94%) and very low organic solventsresidue(DCM and1,4-dioxane were only12ppm and17ppm, respectively). Thecytotoxicity of scaffolds were assessed by Alamar Blue, the results showed a lowtoxicity at the qualified level. The scaffold materials showed a better anticoagulantactivity than the control group through the dynamic blood coagulation experiments.Secondly, the interactions of different nanotopographies with L929and MC3T3-E1on cell adhesion, cell proliferation and protein excretion were investigated. Itturned out that the MC3T3-E1had a best cell adhesion in scaffolds of mixture group,while it was the worst in scaffolds of dioxane group, and the difference wassignificant (P<0.05). The cells were proliferating as the culture time went for5days;while after7days, the numbers of cell decreased in scaffolds of dioxane group, andthere was no significant changes occurred in the dichloromethane group. However,the cells were proliferating slowly in the scaffolds of mixture group at the same time.The total protein excretion amount was detected by Bradford agent, and a highestvalue was obtained in the scaffolds of mixture group, the opposite phenomenon wasobserved in scaffolds of dioxane group. Compared with MC3T3-E1, the L929exhibited an increase on cell adhesion rate and a more protein excretion amount, while a less cell proliferation rate in scaffolds of all three structures. The preliminaryexperimental results showed that the scaffolds of mixture group were benefit for cellgrowth. Based on this result, the effect of adding different amount of fibroin particlesin this scaffolds (PLLA/SF (w/w)=9:1,8:2and7:3) on cell behaviors was examined.The results demonstrated that the material hydrophilic and cellular affinity could beimproved by adding of fibroin particles; furthermore, the cell adhesion of scaffoldstowards to MC3T3-E1was improved. With the increase of culture time, the cell ofgroup including20%fibroin particles could continuously proliferate, while the cellnumbers of group including30%fibroin particles were decreased; however, the cellnumbers of group including10%fibroin particles was not changed significantly. Theadding of fibroin particles in scaffolds could increase the cell protein excretionamount, compared with the group without adding of fibroin particles. On the contraryof MC3T3-E1, the adding of fibroin particles decreased cell adhesion of L929.Besides, the L929grew faster in scaffolds including fibroin particles, and the totalprotein excretion amount was further increased. In conclusion, the group including20%fibroin particles is more suitable for cell growth.Afterward, the potential of in vitro construction of bone tissues were explored bypicking out the scaffolds of dichloromethane group, scaffolds of mixture group andthe scaffolds including20%fibroin particles. Osteoinductive condition was examined.The MC3T3-E1were planted into scaffolds. After being cultured for7,14and21days, the alkaline phosphatase activity was detected by micro plate method, and theosteocalcin and collagen Ι amount were detected by Enzyme Linked ImmunosorbentAssay. Compared to the other two scaffolds, the scaffold of PLLA/SF8:2had ahigher alkaline phosphatase activity, osteocalcin and collagen Ι amount.Finally, in vitro degradation ability of the scaffolds was examined. It showedthat the degradation rate of scaffolds including fibroin particles was faster than thepure PLLA group. In the first2months, the mixture group scaffolds has a lowerdegradation rate of than dichloromethane group scaffolds, while after2months, anopposite phenomenon occurred.