Study On Cytocompatibility Of NPY-PLGA Sustained Releasing Microspheres And Human Adipose-derived Stem Cells

Objective:(1) Human adipose-derived stem cells (hADSCs) were isolated from omental adipose tissue. The cells were cultured and induced to two-line differentiation. Study its characteristics and advantages as an ideal seed cells in adipose tissue engineering;(2) Constructed NPY-PLGA microsphere sustained release system in vitro successfully, and evaluated its sustained release ability by detecting the release characteristics;(3) Investigated the effect of NPY-PLGA sustained release microspheres on toxicity and apoptosis of hADSCs, analyzed cytocompatibility of NPY-PLGA sustained release microspheres, and study the feasibility of NPY-PLGA sustained release microspheres in adipose tissue engineering.Methods:(1) Isolation of hADSCs from omental adipose tissue and two-line differentiation. hADSCs were collected from omental adipose tissue of surgical patients through the method of type I collagenase digestion, filtration, centrifugation, and then went on primary culture. Observed the growth state of the cells. Took the third generation of hADSCs for further experiments. Detected cell proliferation and draw growth curve of hADSCs for several days by cell counting method. hADSCs in logarithmic growth phase were calculated doubling time. Induced hADSCs to adipogenic, osteogenic differentiation with the corresponding inductive culture medium, Oil Red O, Alizarin red staining correspondingly.(2) Constructed NPY-PLGA sustained release microspheres, and investigated ability of microspheres release. Constructed NPY-PLGA sustained release microspheres using ultrasonic emulsification in vitro. Observed morphology and measured the average diameter of microspheres under scanning electron microscopy. Calculated yield rate and detected drug loading rate and encapsulation rate of microsphere. Detected the NPY-PLGA microspheres release capability using ELISA kit for25d.(3) Investigated the effect of NPY-PLGA sustained release microspheres on toxicity and apoptosis of hADSCs in vitro. NPY-PLGA sustained release microspheres is saturated with hADSCs culture medium, detected hADSCs toxicity by MTT, Detected the cells apoptosis by Annexin V-FITC apoptosis kit.Results:(1) hADSCs was obtained successfully from surgical patients omental adipose tissue. Morphology of adherent hADSCs is similar to fibroblasts. hADSCs had strong proliferate ability. Detected cell proliferation and draw growth curve of hADSCs for several days by cell counting method, the curve is S-shaped, logarithmic growth phase the cells’doubling time is45.90h. After induced by adipogenic and osteogenic medium2-3weeks, hADSCs were stained with oil red O, alizarin red respectively. The results were positive.(2) Constructed NPY-PLGA sustained release microspheres successfully. The surface of microspheres was smooth, the sphere size was uniform, the form was full; the average diameter of microsphere was (284.30±36.50)μm; the productivity of microsphere was (20.16±1.45)%, the rate of encapsulation was (11.61±0.81)%, the microspheres drug loading rate was{(2.21±0.09)×10-3}%; Detected the microsphere sustained release capability using ELISA kit. The microspheres could release NPY slowly within25d. The first release rate was10.70%, the cumulative releasing rate of25d was81.80%.(3) Detected the effect of NPY-PLGA sustained release microspheres on toxicity of hADSCs by method of MTT. Toxicity evaluation value were zero. Detected the effect of the microspheres on apoptosis of the cells by Annexin V-FITC apoptosis kit, there was no statistically significant between the experimental group and control group (p>0.05).Conclusions:(1) Fibroblast-like cells could be successfully separated and extracted from omental adipose tissue through primary cell culture technology, hADSCs were identified by morphology and two-lined differentiation. hADSCs were easy to obtain and could passage stably, which could be used as an ideal seed cells for tissue engineering.(2) Constructed NPY-PLGA sustained release microspheres by ultrasonic emulsification in vitro successfully, which also shown a good sustained release ability.(3) NPY-PLGA sustained release microspheres could have good cytocompatibility with hADSCs.