Biocompatibility Of Induced Adipose Derived Stem Cells And Absorbable Gelatin Sponge

ObjectiveTo induced rabbit ADSCs to osteoblasts, and then compounded it withAbsorbable gelatin sponge in vitro, to observed the adhesion, proliferation andthe biological characteristics of ADSCs.To provide experiment base forabsorbable gelatin sponge as an effective carrier of fat stem cells in vitro.Method1.Harvest、culture and expansion of Adipose derived stem cellsADSCs were isolated from rabbit7-day-old, The back of the neck and groinfat pads were isolated and digsted with collagenase Ⅰ. ADSCs were plated byadherent culture and amplified in vitro， to purified the cells by a subsequentpassage.2.Identification of ADSCsTake the third generation of cell population of good condition. To confirmthis cell population were ADSCs by differentiate toward the osteogenic,adipogenic, myogenic, chondrogenic lineages and expressed multiple CDmarker antigens were detectd by flow cytometry.3.Biocompatibility of ADSCs and Absorbable gelatin spongeObserved the primary, the fifth passage, the tenth passage andOsteosarcoma cell, and drawed the cell growth curve.Osteogenic differentiationwas induced by culturing ADSCs for3weeks in osteogenic medium.Toembedded absorbable gelatin sponge by Polylysine before co-culture and thenseeded osteoblasts in absorbable gelatin sponge with pretreatment. The cellsgrowth was measured with MTT assay.This cells were observed under invertedmicroscope and scanning electron microscopy, to observed the adhesion, proliferation and the biological characteristics of ADSCs.To provide experimentbase for absorbable gelatin sponge as an effective carrier of fat stem cells invitro.Results1.Harvest and culture of ADSCsThe ADSCs isolated from rabbits adipose tissue which can rapid growth invitro and fibroblast-like cell growth.The primary ADSCs were observed that alarge number of cell have the same shapes under microscope, such asroundness or type circular.At24hours, a large number of cells transformedgradually into flatteningly and fusiform, which exhibited round or oval. After48hours of culture, obesrved under microscope a large number of cells adherentgrowth, cells exhibited short spindle which were colony-like growth.At7-9days,Cells were covered the90%bottom of the bottle which exhibited fusiform,polygonal and oval. Primary cells contains a large number of red blood cellsand fat cells, all the no-adherent cells were removed through2-3times culturemedium change.2.Identification of ADSCsADSCs were positve expressed CD29and CD44, and negative expressedCD33and CD34. Adipogenic differentiation was induced by culturing ADSCs for2weeks in adipogenic medium,and observed calcium nodules which werestained by Oil Red O. Osteogenic differentiation was induced by culturingADSCs in osteogenic medium3weeks,and extracellular matrix calcification byalizarin red S staining. Chondroinductive differentiation was induced byculturing ADSCs in Chondroinductive medium2weeks, induced ADSCs werestained blue by Toluidine blue staining.3.Biocompatibility of ADSCs and Absorbable gelatin spongeThe oncogenicity potential were detected through comparing the growthcurves of the primary, fivth and thnth passage ADSCs with glioma. Less than10generations ADSCs with no significant difference in expansion（P＞0.05） andno significant tumorigenicity(P＜0.05). The thrth passage of ADSCs wereinduced to osteoblasts and then seeded in absorbable gelatin sponge.Co-culture osteoblasts with absorbable gelatin sponge, which have90% average adherence rate at24h. There was no significant difference betweenosteoblasts seeded in absorbable gelatin sponge with seeded in culture plate(P＞0.05) through MTT assay.The cells began grew At the fourth day and patrs ofcells grew in clumps at seventh day on the surface of absorbable gelatinsponge, and secrete large amounts of cell matrix.ConclusionInduced ADSCs have good Biocompatibility with absorbable gelatinsponge, and absorbable gelatin sponge can be used as biological carrier ofcells.