Study On Biocompatibility Between Growth Factors-coated Electrospun PLCL/fibrinogen Nanofibrous Films And Rabbit Bone Marrow Mesenchymal Stem Cells In Vitro

Objective To explore a method of isolation, culture, purification and identification ofbone marrow-derived mesenchymal stem cells (BMSCs) of rabbit in vitro for the sakeof further study.Methods Bone marrow was harvested from bilateral posterior iliac crest bone of2-month old health New Zealand white rabbit. BMSCs were separated and purified withFicoll separating medium by density gradient centrifugation and adherence culturemethod. The primary and passage cell morphology and growth condition was observedunder inverted phase-contrast microscope. Growth curves of the passage1and passage3rabbit BMSCs were drawn by MTT method and analyze cell growth rhythm. Thesurface antigens of BMSCs, CD29,CD44,CD45,HLA-DR were identified by flowcytometry.Results Primary cultured cells started adhering to plates24hours after seeding andspindle-shaped cells were observed3days after seeding under microscope and growingat a rapid speed. Confluence of cells reached to90%7-8days after seeding. The cellsgrowth curve was S shape. CD29(+),CD44(+),CD45(-) and HLA-DR(-) were detectedby flow cytometry. Conclusion Purified BMSCs of rabbit could be obtained via density gradientcentrifugation and adherence culture method, and BMSCs were proliferated stably andrapidly. BMSCs of3-5generations possessed high activity of growing and proliferatingand were effective to the further study. Objective To investigate the impact of growth factors-coated electrospunPLCL/fibrinogen nanofibrous films on the growth of rabbit bone marrow mesenchymalstem cells(BMSCs).Methods Electrospun nanofibrous films were fabricated frompoly(L-lactide-co-ε-caprolactone)(PLCL) solution(8%w/v) and fibrinogen solution(100mg/ml) with the volume ratio of2:1, and characterized by scanning electronmicroscopy(SEM) and water contact angle analyzer. The nanofibrous films whichsurface modification was performed by immersion into fibronectin solution(20ug/ml)and growth factors solution (VEGF,40ng/ml; bFGF,200ng/ml) sequentially wereexperimental group A films, only by immersion into growth factorssolution(VEGF,40ng/ml; bFGF,200ng/ml) were experimental group B films, andwithout any treatment for the control group. The in vitro release results of growthfactors of experimental group A and B were detected by Enzyme-linked immunosorbentassay (ELISA). The adhesion and proliferation results of rabbit bone marrow mesenchymal stem cells (BMSCs) on different films were detected by MTT assay.Results The average diameter and water contact angle of PLCL/fibrinogen nanofibrouswas305±87nm and (70.21±2.13)°, respectively. Both in experimental group A and B,the growth factors were released in vitro for over10days. Compared to experimentalgroup B and control group, rabbit bone marrow mesenchymal stem cells (BMSCs) inexperimental group A were exhibited highly enhanced adhesion and proliferation (p＜0.05).Conclusions Electrospun PLCL/fibrinogen nanofibrous films, which surface modifiedby fibronectin and growth factors(VEGF, bFGF) sequentially, have good performance ingrowth factors sustained release and cell compatibility aspects and great potentialapplications in vascular tissue engineering field.