聚乳酸-羟基乙酸编织网／胶原-壳聚糖多孔支架促血管化研究

Vascularization of poly-lactic-co-glycolic acid knitted mesh/collagen-chitosan hybrid scaffold

目的观察聚乳酸．羟基乙酸（PLGA）编织网／胶原-壳聚糖多孔支架（PCCS）对血管化的影响，探讨其相关机制。方法（1）采用冷冻-冻干法制备PCCS和胶原-壳聚糖支架（CCS），对比观察PCCS、PLGA编织网及CCS的微观形态及吸水能力。（2）取PCCS与CCS样本，分别植入24只SD大鼠脊柱两侧皮下，按照随机数字表法将大鼠分成3批，于术后1、2、4周分批处死大鼠，采集埋植部位皮肤标本，行组织病理学、免疫组织化学检测；采用实时定最RT—PCR检测其α平滑肌肌动蛋白（α-SMA）与血管内皮生长因子（VEGF）的mRNA表达水平。对数据进行t检验。结果（1）PLGA编织网与胶原-壳聚糖海绵紧密结合，形成的PCCS具有与CCS相类似的三维多孔结构。（2）PCCS的吸水率为（506±15）％，显著高于PLGA编织网的（195±15）％，明显低于CCS的（627±21）％，t值分别为11．9、3．8．P〈0．05或P〈0．001。（3）CCS4周左右完成支架全层的组织长人，PCCS仅用2周完成这一过程且形成的新生胶原更加丰富、分布均匀。（4）随着埋植时间的延长，2种支架的血管逐渐由支架剧围向支架内部长人。术后1、2、4周，PCCS中血管计数依次为（10．7±3．2）、（18．6±2．1）、（30．3±4．5）条／mm2，明昆高于CCS的（5．4±0．9）、（10．8±4．2）、（23．6±1．7）条／mm2，t值分别为4．6、4．4和4．5，P值均小于0．01。（5）术后各时相点PCCS中α-SMA与VEGF的mRNA表达水平均高于CCS（t值分别为1．26、1．63、2．17与5．52、2．07、1．78，P值均小于0．01）。结论PCCS能够快速诱导血管长入并促进血管成熟，其中支架的机械支撑作用与三维多孔结构具有重要协同作用。

Objective To investigate the effects of poly-lactic-co-glycolic acid （PLGA） knitted mesh/collagen-chitosan hybrid scaffold （PCCS） on angiogenesis, and to explore the relative mechanisms. Methods PLGA knitted mesh was integrated into collagen-chitosan scaffold （CCS） to construct PCCS with freeze-lyophilizing method, and CCS was made with the same method. The characteristics of morphology and water absorbing capacity among PCCS, PLGA knitted mesh, and CCS were compared in vitro. PCCS anti CCS was respectively implanted into subcutaneous tissue of back on both sides in 24 SD rats, and the tissue specimens were harvested at post operation week （POW） 1 , 2, and 4 according to the random number table to evaluate the level of angiogenesis by histopatho/ogical and immunohistochemical examinations. The expression levels of alpha smooth muscle actin （α-SMA） and vascular endothelial growth factor （VEGF） mRNA were examined by real-time quantitative RT-PCR. Data were processed with t test. Results （ 1 ） PLGA knitted mesh was closely integrated with sponge of collagen-ehitosan in PCCS, anti the porous structure of PCCS was similar to that of CCS. （2） Compared with that of PCCS [（506± 15）% ], the water absorbing rate of CCS and PLGA knitted mesh was respectively increased and decreased [ （627 ± 21 ） % , （ 195 ± 15）% , witht value respectively 3.8, 11.9, P 〈0.05 orP 〈0.001 ]. （3） The scaffolds were filled with newly formed tissue in CCS at POW 4, while those in PCCS were observed at POW 2 with more homogeneous and abundant collagen. （4） Blood vessels could be induced, and they grew into scaffolds along with prolongation of implantation time in PCCS and CCS. The number of mature blood vessels in PCCS at POW 1 , 2, 4 [ （ 10.7 ± 3.2） , （ 18.6 ± 2.1 ） , and （30.3 ± 4.5） branches per square centimeter] was respectively higher than that in CCS [（5.4±0.9）, （10.8±4.2）, and （23.6±1.7） branches per square centimeter, witht value respectively 4.6, 4. 4, 4.5, P values all below 0.01 ]. （5） The expression levels of ct-SMA and VEGF mRNA in PCCS at POW 1, 2, 4 were significantly higher than those in the CCS （with t α-SMA value respectively 1.26, 1. 63, 2. 17, with tVEGF value respectively 5.52, 2.07, 1.78, P values all below 0.01 ）. Conclusions PCCS is able to induce the ingrowth of blood vessels rapidly and promote their maturity. The mechanical properties and microstructures of scaffolds play synergistic role in the process of angiogenesis.