80 Journal of Tissue Engineering and Reconstructive Surgery June 2011 Vol.7 No.2 doi:10.3969/j.issn.1673-0364.2011.02.005 PLG - [Poly(DL-lactic-co-glycolic acid)plg](l G=75 25) PLG MTT PLG S-100 (P>0.05) PLG - Q813.1+1R318.08 1673-0364(2011)02-0080-05 iocompatibility of PLG with Olfactory Ensheathing ells in Rat UI Ying UI Zhiming XU Guanhua WNG Lingling SUN Yuyu HU Jingzhe. Department of Spine Surgery Second ffiliated Hospital of Nantong University Nantong 226001 hina. orresponding author: UI Zhiming (E-mail:czmspine@163.com). bstract Objective To explore the biocompatibility of Poly(DL-lactic-co-glycolic acid) (PLG) (L G=75 25) with olfactory ensheathing cells (OEs) in rat. Methods The purified OEs were seeded on the PLG membrane (PLG group) and on the columns coated Poly-L-Lysine (control group). The adhesion and viability of OEs were observed by inverted microscope and scanning electron microscopy. MTT method and the computer image statistical software were used to determinate the survival and proliferation of OEs. Results OEs grew well on PLG membrane. There were no significant differences in the activity of OEs. The number of S-100 positive cells the area of the cell bodies and the perimeter of the cell between two groups (P>0.05). onclusion The PLG biomaterial has good biocompatibility with rat OEs. It could be an ideal tissue engineered scaffold material in the repair of spinal cord injury. Key words Olfactory ensheathing cells; Poly(DL-lactic-co-glycolic acid); iocompatibility; Spinal cord injury 1-1.1 [poly(dl-lactic-co-glycolic acid)plg] 1~3 d SD ( (L) );PLG(L G=75 25IV=2.0 dl/g (G) );DMEM/F12 [1] (Olfactory ensheathing (Gibco );NGFRp75 (Santa ruz cellsoes) );Hoechst 33342 S-100 (Sigma );lexa [2-4] fluor 568 lexa fluor 488(Invitrogen ) OEs 1.2 PLG L G=75 25 PLG PLG 10%(w/v) PLG OEs : (R2007027) (S2009014) : (E-mail:czmspine@163.com) :226001 0.5 mm PLG 70% 3
2011 4 7 2 81 PLG 2 cm 2 cm ph 7.2 PS 37 PLG M 1 M 2 37 M 1-M 2 25% ~49% 50% ~74% 75% ~ W= 100% M 1 99% 100% 0 RGR 1.3 OEs 1.7 1~3 d SD 200 DMEM/F12 801 2 200 S-100 1 mm 3 0.125% S-100 O 2 37 20 min ( ) 3 10% (FS) ± 5 min 400 SPSS13.0 P<0.05 1 000 r/min 10 min 2 10%FS 100 U/mL 100 mg/ml 2.1 PLG 37 5%O 2 PLG PS 18 h 36 h ( 1) 2 8 48 h 8 12 14% PLG 7 OEs Hoechst 33342 S- ( 2) 100 NGFRp75 2.2 OEs Hoechst 33342 S-100 NGFRp75 1.4 OEs PLG OEs 91% 1 10 6 cells/ml OEs ( 3) PLG 2.3 OEs PLG (PLL) OEs PLG 37 5%O 2 3 12 h 80% OEs 1 d 3 d 5 d 7 d PLG 1 d 1.5 3 1 3 5 7 4% 5 30 minps 3 1 h NGFRp75 S- 7 100 4 PS 3 lexa fluor 568 lexa fluor 488 IgG 2 h 1.6 OEs PLG 1 d 3 d 5 d 7 d 5 mg/ml MTT 570 nm OD (RGR) :0% 1%~24% ( 4)
82 Journal of Tissue Engineering and Reconstructive Surgery June 2011 Vol.7 No.2 2.4 OEs PLG OD 1 PLG OEs (%) 16 14 12 10 8 6 4 2 0 Fig.1 2 4 6 8 10 ( ) 1 PLG 12 Degraded curve of weight loss of PLG membrane in 12 weeks in vitro 12 14 2.5 S-100 ( ) (P>0.05)( 2 5) D : ;: 4 ;: 8 ;D: 12 :efore degradation; : 4 weeks after degradation; : 8 weeks after degradation; D: 12 weeks after degradation 2 PLG (1 500 ) Fig.2 SEM observation of degradation of PLG membrane (1 500 ) :S-100 :NGFRp75 :Hoechst33342 : Immuno-fluorescent staining of S-100 special antibody : Immuno-fluorescent staining of NGFRp75 : Immuno-fluorescent staining of Hoechst33342 special antibody 3 7 d OEs(200 ) E Fig.3 OEs 7 days after culture (200 ) D F G H : 5 d OEsPLG : 5 d OEs : OEs 5 days after co-culture PLG group : OEs 5 days after co-culture control group 5 (200 ) Fig.5 Immuno-fluorescent staining observation (200 ) 4 (1 500 ):PLG 1 d() 3 d() 5 d() 7 d(d); 1 d(e) 3 d(f) 5 d(g) 7 d(h) Fig.4 SEM observation (1 500 ): PLG group 1 d () 3 d() 5 d () 7 d (D) after culture; control group 1 d (E) 3 d (F) 5 d (G) 7 d (H) after culture
2011 4 7 2 83 Table 2 Table 1 1 OD (x±sn=6) OD values of two groups in different time (x±sn=6) 1 d 3 d 5 d 7 d 0.287±0.024 0.353±0.029 0.471±0.029 0.647±0.033 ontrol group(od value) PLG PLG group(od value) 0.274±0.022 0.320±0.027 0.422±0.031 0.591±0.034 RGR 95.4% 90.7% 88.1% 89.4% ontrol group PLG PLG group 2 S-100 (x±sn=12) S-100 positive cell count cell bodies area and cell circumference of two groups (x±s n=12) *P=0.267 **P=0.228 ***P=0.141 S-100 (μm 2 ) (μm) S-100 positive cell count cell bodies area (μm 2 ) cell circumference (μm) 21.53±3.81* 368.43±22.49** 135.39±13.66*** 19.88±3.26* 357.74±19.63** 127.42±11.85*** 3 75 25 PLG 2 8 Wu OEs [12] Raisman Wu OEs PLG ; [5-6] ph [7-8] PLG (L) (G) PLG L G 75 25 PLG 8 [9-10] Moore [11] L G=85 15 PLG (PLL) 1 [13] Teng [6] PLG PLL OEs PLG PLG OEs PLG OEs G L S-100 PLG G PLG 75 25 OEs
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