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1 80 Journal of Tissue Engineering and Reconstructive Surgery June 2011 Vol.7 No.2 doi: /j.issn PLG - [Poly(DL-lactic-co-glycolic acid)plg](l G=75 25) PLG MTT PLG S-100 (P>0.05) PLG - Q R (2011) 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 hina. orresponding author: UI Zhiming ( 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 [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 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 : (R ) (S ) : ( czmspine@163.com) : mm PLG 70% 3

2 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/F S mm % S-100 O min ( ) 3 10% (FS) ± 5 min 400 SPSS13.0 P< 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) h % PLG 7 OEs Hoechst S- ( 2) 100 NGFRp OEs Hoechst S-100 NGFRp OEs PLG OEs 91% cells/ml OEs ( 3) PLG 2.3 OEs PLG (PLL) OEs PLG 37 5%O h 80% OEs 1 d 3 d 5 d 7 d PLG 1 d % 5 30 minps 3 1 h NGFRp75 S 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.

3 82 Journal of Tissue Engineering and Reconstructive Surgery June 2011 Vol.7 No OEs PLG OD 1 PLG OEs (%) Fig ( ) 1 PLG 12 Degraded curve of weight loss of PLG membrane in 12 weeks in vitro 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

4 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± ± ± ±0.033 ontrol group(od value) PLG PLG group(od value) 0.274± ± ± ±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* ±22.49** ±13.66*** 19.88±3.26* ±19.63** ±11.85*** PLG 2 8 Wu OEs [12] Raisman Wu OEs PLG ; [5-6] ph [7-8] PLG (L) (G) PLG L G 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 OEs

5 84 Journal of Tissue Engineering and Reconstructive Surgery June 2011 Vol.7 No.2 OEs-PLG PLG MTT Neurol (2): [5] Potter W Kalil RE Kao WJ. iomimetic material systems for neural OEs PLG progenitor cell-based therapy[j]. Front iosci200813: %~99% [6] Teng YD Lavik E Qu X et al. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold PLG OEs seeded with neural stem cells [J]. Proc Natl cad Sci US (5): [7] Novikova LN Kellerth JO Novikov LN. iopolymers and biodegradable OEs PLG OEs PLG [8] smart implants for tissue regeneration after spinal cord injury [J]. urr Opin Neurol200316(6): Krych J Rooney GE hen et al. Relationship between scaffold channel diameter and number of regenerating axons in the transected rat spinal cord [J]. cta iomaterialia20095(7): [9] Takezawa T. strategy for the development of tissue engineering scaffolds that regulate cell behavior [J]. iomaterials200324(13): [10] Qiu LY Zhu KJ. Novel biodegradable blends of poly [bis(glycineethyl [1] Di Toro R etti V Spampinato S. iocompatibility and integrinmediated adhesion of human osteoblasts to poly(dl-lactide-coglycolide) copolymers [J]. Eur J Pharm Sci200421(2-3): ester) phosphazene] and polyesters or polyanhydrides: compatibility and biodegradable characteristics [J]. Polym Int200049(11): [2] Willerth SM Sakiyama-Elbert SE. ell therapy for spinal cord regeneration [J]. dv Drug Deliver Rev200860(2): [11] Moore MJ Friedman J Lewellyn E et al. Multiple-channel scaffolds to promote spinal cord axon regeneration [J]. iomaterials [3] Franssen EH de ree FM Verhaagen J. Olfactory ensheathing glia: their contribution to primary olfactory nervous system regeneration and their regenerative potential following transplantation into the injured spinal cord [J]. rain Res Rev200756(1): [12] (3): Wu L Ding JD. In vitro degradation of three-dimensional porous poly(dl-lactide-co-glycolide) scaffolds for tissue engineering [J]. iomaterials200425(27): [4] Guest JD Herrera L Margitich I et al. Xenografts of expanded primate olfactory ensheathing glia support transient behavioral recovery that is independent of serotonergic or corticospinal axonal regeneration in nude rats following spinal cord transection [J]. Exp [13] Kim J Williams J. Rapid prototyping of patterned poly-l-lysine microstructures [J]. onf Proc IEEE Eng Med iol Soc20061: ( : ; : )!!!!!!!!!!!!!!!!!!!!!!! ( 69 ) iomaterials200930(4): [8]. [J]. lood (9): [J]. [14] Koike N Fukumura D Gralla O et al. Tissue engineering: creation 20095(2): of long-lasting blood vessels [J]. Nature (6979): [9]. [15] Ying X Zhe X Hellem S et al. Endothelial cells influence the [J] (1): [10] Lokmic Z Stillaert F Morrison W et al. n arteriovenous loop in a protected space generates a permanent highly vascular tissueengineered construct [J]. FSE J21(2): [11] Kaigler D Krebsbach PH Polverini PJ et al. Role of vascular endothelial growth factor in bone marrow stromal cell modulation of endothelial cells [J]. Tissue Eng20039(1): [12] Villars F ordenave L areille R et al. Effect of human endothelial cells on human bone marrow stromal cell phenotype: role of VEGF [J]. J ell iochem200079(4): [13] u P Tam J Fukumura D et al. one marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature osteogenic potential of bone marrow stromal cells [J]. iomed Eng Online20098:34. [16] ouletreau PJ Warren SM Spector J et al. Hypoxia and VEGF up-regulate MP-2 mrn and protein expression in microvascular endothelial cells: implications for fracture healing [J]. Plastic Reconstru Surg (7): [17] Kaigler D Krebsbach PH West ER et al. Endothelial cell modulation of bone marrow stromal cell osteogenic potential [J]. FSE J (6): ( : ; : )

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