Influences Of Gravity Vector And Chemotaxis On ASCs Adhesion And Proliferation Onto PLGA Electrospinning Membrane At Early Stage

Objectives To observe the characteristics of adhesion and proliferation in vitro of adipose-derived stem cells (ASCs) subjected to various gravity vectors with/without the chemotactic factors of platelet-derived growth factor (PDGF) on the electrospun poly lactic-co-glycolic acid (PLGA) membranes, and to explore the effects of gravity factors and chemotactic factor on the cell adhesion and function of ASCs onto the tissue engineering scaffold.Materials and methods1. Electrospinning preparation of PLGA membranes24ml Trichloromethane (TCM, analytical reagent)and6ml N,N-Dimethylforrmamide (DMF,analytical reagent)mixed (volume ratio=8:2), then5.4g PLGA (intrinsic viscosity0.76dl/g, weight-average molecular weight80,000Da) was dissolved into this mixture to form18%electrospun solution. The electrospinning of such solution was performed in a14KV static electric field with the rate of1ml/h and distance of12cm. The final PLGA membrane is of600μm in thickness,26.0×19.0cm2maximal available area,0.172g/cm3in density and5.1g in weight. Standard round-shaped specimens were then cut from this membrane in diameter of4.5mm, and reserved for the experiment after24hours Cobalt-60radiosterilization.2.Harvest of ASCs10ml fresh human buccal fat pad adipose tissues retrieved from face contour surgery were cut into1mm’particles and digested with type I collagenase. ASCs were then harvested with gravity gradient centrifugation method, and cultured in complete dulbecco’s modified eagle medium under the condition of37℃,5%CO2and saturated humidity for14days. Medium exchange was performed every three days and ASCs were passaged as the cell contact up to80%till the fifth passage (P5), then the ASCs were transferred to-80℃conservation for further experiment.3.Grouping of the experiment Resuscitated P5ASCs were cultured following forementioned protocol and passaged to the sixth as the cell contact up to80%. After being digested by0.25%trypsase, the P6ASCs were suspended in DMEM to3×104/ml cell concentration. Each100μl(3000cells) of ASCs suspension was then transferred to96-well plate for72wells that devided into4groups (18wells each group):(1)PDGF/gravity (+):the electrospun PLGA membranes contained0.01ng PDGF was placed on bottom of the well (ASCs floating over the membranes);(2)PBS/gravity (+):the electrospun PLGA membranes contained1μl blank Phosphate Buffered Saline (PBS) was placed on bottom of the well (ASCs floating over the membranes);(3)PDGF/gravity (-):the electrospun PLGA membranes contained O.Olng PDGF was placed on top of the well (ASCs under the membranes);(4)PBS/gravity (-):the electrospun PLGA membranes contained1μl blank PBS was placed on top of the well (ASCs under the membranes).4.Test and analysis The sample-loaded96-well plate was cultured under the condition of37℃,5%CO2and saturated humidity.10μl Alamar Blue reagent was added continuously into each3wells out of all the four groups at time intervals of0,1,3,5,8,11days respectively, and another24hours co-incubation was then performed. The optical density (OD) value of the culture filtrate was measured and recorded at570nm and600nm wave length. Relative cell number and proliferative activities were calculated as percentage reduction of Alamar Blue. Thereafter, to analyse chemotaxis of ASCs to adhesion onto the electrospun PLGA membranes by PDGF, furthermore, the interaction between gravity factor and PDGF.Data analysis was processed by SPSS20.0software, and P<0.05was considered as statistically significant different.Results1. In regard of influence of gravity vector, being loaded with PDGF, the decrease percentage of Alamar Blue of gravity (+) group was significantly higher than that of gravity (-) group on day1,2,4,6,9and12. In two control groups in which PBS was loaded instead of PDGF,showed the similar results. Therefore, positive gravity vector could be stimulative factor for adhesion and proliferation of ASCs on the electrospun PLGA membranes than negative gravity vector.2. In regard of chemotactic effect of PDGF. under positive gravity vector, the decrease percentage of Alamar Blue of PDGF and PBS group on day1,2,4were approximately equal, while that of PDGF group was significantly higher than that of PBS group on day6,9and12. In two control groups in which the gravity vector presented negatively, significant difference was noticed on day1,2,4,6,9and12. Therefore, the presence of PDGF could be another stimulative factor for adhesion and proliferation of ASCs on the electrospun PLGA membranes. Furthermore, along with the extension of experimental time interval, the difference became more significant proving the dramatic chemotaxis effect of PDGF.3. Considering the temporal-spatial changing tendency of ASCs proliferative activities on electrospun PLGA membrane, the cells began adhesion onto the scaffold, with vigorous proliferation and function activities, obvious difference was observed among all the four groups on day6,9and12. Gravity vector and PDGF chemotactic effect, had obvious influence on cell adhesion and proliferation in tissue engineering, and with time prolonged, the interactive effect of the two factors had superposition effect on ASCs.Conclusions1. The electrospun PLGA membranes which prepared by electrospun technology, had high porosity and porous, the structure of scaffold fibre was similar to extracellular matrix, in favour of planting, adhesion and proliferation for ASCs.2. Cytokines PDGF efficiently induced ASCs chemotactic aggregation, and significantly improve the adhesion and proliferation of ASCs onto the electrospun PLGA membranes.3.The gravity vector factor can obviously effect the adhesion and proliferation of ASCs on the electrospun PLGA membranes, and such influence could be observed throughout our experiment.4. The adhesion, planting and proliferation activities of stem on the scaffold material could be affected obviously by various factors (gravity, factor etc.).With time prolonged, the interactive effect of the factors had superposition effect on cells.5.Application of chemotactic chemokines could effectively promoted the adhesion and proliferation of seed cells onto the scaffold material in early stage, and helped to compensate the negative influence of unfavorable gravity vector obviously, improved efficiency of tissue engineering.