Effects Of Liquid Crystal Polymer Cholesterol-graft-lactic Acid On Fibroblast Phenotypic Transformation And Apoptosis

Objective:Evaluating the effects of biodegradable polymer material cholesterol oligo-graft-lactic acid?CLA?on fibroblast adhesion,proliferation and phenotypic transformation.Then,the correlation between CLA degradation and liquid crystal structure change and fibroblast apoptosis were also been investigated.Methods:?1?The CLA oligomer was synthetized by ring-opening polymerization of cholesterol and lactide,with weight ratio of 1:1.5,1:2.5,and 1:3.5 and named as CLA_1:1.5,CLA_1:2.5 and CLA_1:3.5.The polymer structure was identified by infrared spectroscopy and hydrogen-nuclear magnetic resonance.The liquid crystal state,phase transition temperature range and relative molecular weight were determined by polarizing microscope,differential scanning calorimetry and gel permeation chromatography.?2?Fibroblasts of NIH 3T3 were respectively seeded on matrix materials of bovine tendon collagen,polylactic acid?PLA?and CLAs with different cholesterol grafting degree,with blank culture plate as control.The cell adhension and proliferation were analysed by CCK-8 counting kit.The expression of?-smooth muscle actin??-SMA?,the index of fibroblast phenotypic transformation to myofibroblast,was detected by the western blot and the immunofluorescence stain.?3?CLAs with different cholesterol grafting degree were incubated by phosphate buffer.The differential scanning calorimetry and polarizing microscope were used to analyse the relationship between polymer degradation and liquid crystal state and phase-transition temperature.The effects of polymer degradation of fibroblast apoptosis were analyzed by TUNEL and Annexin-PI stain.Results:?1?The polymers of CLA_1:1.5,CLA_1:2.5 and CLA_1:3.5 exhibited yellow and solid appearance,relative molecular weight of 793±140,1320±123 and 1366±146,and characteristic birefringence in polarized light at 37?,which indicated the liquid crystal state.The bovine tendon collagen also exhibited liquid crystal conformation,however PLA did not.?2?When cultured for 4 h,CLA_1:1.5 and CLA_1:2.5 can significantly promoted FB adhesion compared to control and PLA group?P<0.05?,but showed no significant difference with collagen group?P>0.05?.And CLA_1:3.5showed no significant difference with control?P>0.05?.When cultured for 24 h,CLA_1:1.5,CLA_1:2.5 and CLA_1:3.5 can significantly promoted FB proliferation compared to control?P<0.01?,but showed no significant difference with collagen group?P>0.05?.There was no significant difference in three CLA groups?P>0.05?.There was no significant difference iná-SMA expression in all groups?P>0.05?.When cultured for 48 and 72 h,FB proliferation of CLA_1:3.5 showed no significant difference compared with control and PLA?P>0.05?.but CLA_1:1.5 and CLA_1:2.5 can promoted FB proliferation compared to control?P<0.01?and showed no significant difference with collagen?P>0.05?.In addition,á-SMA expression of CLA is higher than control and PLA?P<0.05?but showed no significant difference with collagen group?P>0.05?.There is no significant difference in three CLA groups?P>0.05?.?3?Polarizing microscope was used to observe CLA liquid crystal structure change when they were incubated in PBS buffer.CLA_1:1.5 and CLA_1:2.5's liquid crystal phase were disappeared when incubated for 72 h.However,CLA_1:3.5 membrane can stayed its liquid crystal phase for 96 h because of its higher relative molecular mass.TUNEL and Annexin-PI stain were used to analyse the effect of CLA liquid crystal structure change on fibroblast apoptosis.Incubated CLA_1:3.5 can promoted FB apoptosis compared to control and PLA groups?P<0.05?.Apoptosis was positively correlated with incubated time?P<0.05?.CLA_1:1.5 and CLA_1:2.5 which incubated within PBS buffer for 24,48,72 h can promoted FB apoptosis?P<0.05?.But they showed no effect on FB apoptosis after incubating within PBS buffer for 96 h?P>0.05?.In addition,fibroblast apoptosis was positively correlated with cholesterol ratio at any time?P<0.05?.Conclusions:CLA which is prepared by ring-opening polymerization of lactideby and cholesterol is liquid crystal;its liquid crystal structure can promote fibroblast adhesion,proliferation and differentiation;CLA's liquid crystal structure disappears along with CLA degradation and it can induce fibroblast apoptosis.This paper offers a new standpoint of degradable liquid crystal polymer for the treatment and prevention of hypertrophic scar which deserves more research.