摘要
目的:制备猪小肠粘膜下层(SIS),将其作为组织工程韧带的支架材料,应用同基双功能交联剂SS-PEG3400-SS与重组人骨形态发生蛋白-2(BMP-2)进行共价偶联,并随后与SIS进行共价交联反应,以研究经过共价偶联固化了BMP-2的SIS在缓慢降解的同时,是否可以随着其降解而长时间自发释放BMP-2,从而持续刺激小鼠胚胎成骨细胞株(MC3T3-E1)向成骨细胞定向分化。
方法:将BMP-2与同基双功能交联剂SS-PEG3400-SS进行共价交联,然后将交联后的产物与制备的SIS进行共价偶联,从而间接将BMP-2固化在SIS上。用透射电镜对交联后的SIS进行形态学观察,并评估MC3T3-E1细胞在与多种不同SIS上的增殖及定向分化情况。同时比较与BMP-2共价偶联后的SIS与未与BMP-2共价偶联的SIS上钙结节的数量及碱性磷酸酶活力值,结果采用SPSS11.0软件进行统计学分析。
结果:与未修饰的BMP-2相比,共价修饰后的BMP-2仍具有生物活性。通过将交联剂SS-PEG3400-SS与主要成分为胶原的SIS进行交联,可减慢SIS在体外的降解速度,并且共价交联的BMP-2可随着SIS的降解而长时间释放,从而能持续刺激MC3T3-E1细胞向成骨方向分化。
结论:BMP-2通过同基双功能交联剂SS-PEG3400-SS与SIS共价偶联。共价偶联了BMP-2的SIS能更好刺激与其复合培养的MC3T3-E1细胞向成骨方向定向分化。
Objectives: To prepare porcine small intestine submucosa(SIS) for the scaffold of tissue engineering ligament,and using the homobifunctional cross-linking agent Disuccinimidyldisuccinatepolyethyleneglycol(SS-PEG3400-SS)for both the covalent coupling of the bone morphogenetic proteins-2(BMP-2) to and cross-linking of SIS. And the goal is to evaluate whether the covalent bound BMP-2 will be released simultaneously with the degradation of SIS,and which will lead to the MC3T3-E1 cell directionally differentiate to osteoblast.
Methods: Using the homobifunctional cross-linking agent SS-PEG3400-SS to covalently couple of BMP-2, and the conjugated BMP-2 molecules were then allowed to react with SIS for the immobilisation reaction , which leading to covalently immobilise the BMP-2 within SIS. Transmission electron microscope(TEM) were used to observe the morphous of the cross-linked SIS and evaluate the proliferation and differentiation of MC3T3-E1 cells which exposed to the differently modified and loaded SIS. Comparing to the quantity of calcium nodule and alkaline phosphatase (AKP) in per piece of differently modified and loaded SIS, then statistical analysis was performed by using the software package SPSS11.0.
Results The conjugates (BMP-2)-PEG still have a bioactivity as compared to the non-modified BMP-2. The crosslinking of SIS through corss-linking agent SS-PEG3400-SS stabilize the SIS and slow down its degradation in vitro, and covalent bound BMP-2 can be released persistently and simultaneously with the degradation of the SIS, which can lead to the MC3T3-E1 cell directionally differentiate to osteoblast.
Conclusion: BMP-2 can covalently link to the SIS through the cross-linking agent. After covalently immobilising the BMP-2 within SIS, the directional differentiation to osteoblast of MC3T3-E1 cells which exposed to the modified SIS can be observed.
引文
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