Preparation Of The SF/COL/PLCL Electrospinning Nanofibrous Scaffolds And Study Its Cytocompatibility

Objective Preparation of the SF/COL/PLCL nanofiber scaffold and study its cytocompatibility by use human umbilical cord blood mesenchymal stem cells.Methods1.40-60ml cord blood was collected under sterile conditions, and heparin was used for anti-freezing.The blood samples were isolated by density gradient centrifugation. White layer of mononuclear cells were carefully drew.Then the cell concentration was adusted to1×106/ml,20%fetal bovine serum DMEM/F12medium was added for primary culture, the whole medium was changed after5days, and then the medium was changed every3days. Cells were observed after the adherence and proliferation.Trypsin was used for the digestion after the cells were fused to80%-90%,and then cells were passaged in the ratio of1to2.2. Pupa cocoon was placed into100℃0.5w/v%Na2CO3solution. Then the pupa cocoon was boiled in the Na2CO3solution three times,30minutes each time. And the pupa cocoon was washed well by distilled water to remove the sericin. The degummed silk was dried and was dissolved in CaCl2:C2H5OH:H2O=1:2:8(molar ratio))at70℃for1hour. The cooled pupa cocoon was placed in a dialysis bag for three days, and then filtered, freeze and dried respectively to give pure silk fibroin. Pure SF and COL mixed in the ratio of70:30were dissolved in HFIP, and then8%blend solution was obtained. The blend solution was mixed with PLCL in the ratio of100:0,70:30,50:50,30:70,0:100respectively. Then they were dissolved in HFIP to obtaine ablend spinning solution with a mass ratio of8%. The electrospun scaffold was prepared under a certain condition (voltage of15Kv, jet velocity of1.5ml/h, receiving distance of12cm). Morphology was observed by using SEM and X-ray diffraction was used to examine the crystallinity of the samples.then the mechanical properties were tested and the thermal stability was analysed.3. The third generation of umbilical cord blood mesenchymal stem cells was seeded on silk fibroin/collagen/polycaprolactone nanofiber scaffold. Inverted microscope was used to observe the adhesion, growth and proliferation abilities of the cells. MTT assay was used to analyse the effect of the scaffold to the proliferation of the cells cultured after1,3,5,7days respectively. SEM was used to observe the growth and proliferation of the cells on the surface of the scaffold after cultured5,7,9, days respectively.Results1. After primary cultured after5-7days, the adherent HUCB-MSCs were in a spindle formation of fiber-like cells. The cell morphology is similar to the bone marrow mesenchymal stem cells after cells were cultured to the second generation. The cells showed in a homogeneous fusiform and arranged in alignment.2. With the increase of PLCL content, the average diameter of the fiber scaffold increased. Mechanical properties of the scaffold increased gradually with in increase of PLCL content.3. The proliferation of hUCBMSCs in experimental group was excessed in that of control group. The pure SF/COL and SF/COL/PLCL nanofiber scaffold with different mass ratio can promote cell growth well. Especially in the ratio of SF/COL/PLCL is30:70, which is benifical for the growth of cells.Conclusion The umbilical cord blood mesenchymal stem cells can grow and proliferation in the nano fiber scaffold, this kind of scaffolds material have good cytocompatibility and scaffold performance testing results show that it have good physical properties, and are expected to be as a kind of new scaffold materials.