表面改性的PHBHHx的体外相容性研究
|
摘要
目的
动脉粥样硬化性疾病(AS)是严重危害人类健康的一大类疾病,在发达国家被称为“头号杀手”,是世界范围的主要死亡原因之一。资料显示,1999年全世界死亡人数约5860万.其中因冠心病死亡人数高达860万,位居死因第1位。我国动脉粥样硬化性疾病发病虽没有西方国家那么高,但正呈上升趋势发展。Mustardt等指出,动脉粥样硬化心脏病是导致发病和死亡的原因之一,这种疾病使得动脉管径狭窄,最终使血管堵塞。在此背景下,血管支架因其独特的成型性和生物相容性成为治疗心血管疾病的重要技术,近年来在国内外已被广泛应用。
理想的血管支架应具有以下特征：(1)良好的生物相容性,尤其是血液相容性,即非致血栓性和无免疫原性；(2)适当的机械性能,包括适当的强度和顺应性；(3)适当的生理性能,对神经或化学刺激能做出较好收缩／舒张反应；(4)生产周期短、成本低,而且规格和尺寸齐全能满足临床需要。
据报道,全世界每年有300万人接受心血管支架置人手术,但其置入术后血管再狭窄问题却始终未能得到彻底解决。人们进行了多方面的尝试和努力,发现内皮细胞完整性的破坏及功能紊乱与支架植入后血管再狭窄有着密切的关系,提出将改善内皮细胞(endothelial cells, EC)功能及完整性的恢复作为预防再狭窄的新策略。
内皮细胞不仅是介于血管壁和血液之间的屏障结构。而且是体内一种代谢十分活跃的细胞,能合成和分泌多种生物活性物质,如分泌一氧化氮、前列环素等活性物质,可有效防止血栓形成,维持血管收缩、舒张,凝血与抗凝血等动态平衡,在血管腔的表面形成一抗凝血和抗血栓系统,另外内皮细胞同血细胞一样表面带负电荷,因而具有抗血小板聚集、防止血液凝固和血栓形成的作用,从而保持血液的正常流动和血管的长通畅。人工血管与自体血管的主要区别是无内皮细胞衬里,所以如何使人工血管内皮化,显得尤为重要。
总之,血管内支架内皮化是解决血管内支架置人后血栓形成和提高远期通畅率很有前途的方法之一,通过从细胞和分子水平深入研究材料与特定细胞、组织之间的表面／界面作用,并研究材料学因素和生物化学因素及二者相互作用对细胞的黏附、生长、增殖、分化、基因表达和调控、生理功能的影响因素和规律,进而阐明生物医学材料与目标物质之间的分子识别规律,进而加强血管内支架体外内皮化的基础研究将为其临床应用效果的改善成为可能。
多羟基抗生素类(PHAs)是一类可以由很多种类微生物自然产生的,具有良好生物相容性的生物可降解的共聚物。在PHAs家族中第一个,也是研究最深入的就是具有高结晶性的3-羟基丁酸聚合物,即PHB。低分子量的PHB在血液中的终产物是3-羟基丁酸,一种普通的代谢产物,并且在高级动物的试验中也证明,PHB移植物是无毒的。然而,PHB的脆性和低力学强度明显限制了它的广泛应用。因此,PHA家族中具有更好机械学性能和热力学稳定性的材料,诸如3-羟基丁酸与3-羟基己酸共聚酯(PHBHHx)便成为了更有实际应用价值的材料。目前,体外试验已证明了PHBHHx对一些细胞的生物相容性非常好,如软骨细胞,骨髓间充质干细胞和成纤维细胞。考虑到通过改变其组成的不同组分可以改变它的力学特性的特点,相信PHBHHx一定能更好的满足力学特性要求高的骨组织工程和弹性要求好的血管组织工程的要求。本实验PHBHHx材料经NaOH处理及添加了不同比例的中药涂层改性后,采用人脐静脉内皮细胞、大鼠间充质干细胞与其进行复合培养后,利用形态学、免疫细胞化学、免疫荧光细胞化学以及细胞生长活力等多个指标进行分析,从而探讨表面改性PHBHHx在将来心血管支架应用中的潜在优势,进一步促进经过表面改性的PHBHHx在临床的早日应用。
方法
体重100g雄性wistar大鼠脱颈椎处死,通过全骨髓培养法获得相对均质的BMSCs,细胞爬片,固定,进行CD44、CD 106免疫荧光染色鉴定。取产后4h内的健康新生儿脐带,通过胰酶消化法,获得相对均质的HUVECs,细胞爬片,固定,进行vWF、FLK-1免疫化学染色鉴定。用人脐静脉内皮细胞、大鼠间充质干细胞与表面改性的PHBHHx进行复合培养后,利用扫描电镜观察、免疫荧光染色、中性红染色及MTT法等多个指标进行分析其体外相容性。
结果
一、HUVECs的分离、培养及鉴定
原代培养3d后,HUVECs汇合成单层细胞。传代培养的HUVECs 30min即贴壁,2-3d融合成单层。形态学观察倒置显微镜下内皮细胞呈梭形或多边形,大小均匀。细胞生长融合形成单层后,呈典型的鹅卵石或铺路石镶嵌状排列生长,并有接触抑制现象,具有内皮细胞特征。vWF、FLK-1免疫化学染色显可见胞质内棕黄色颗粒,核周密集,而对照组内未见着色。
二、MSCs的分离、培养及鉴定
BMSCs原代培养接种后12h可见少数细胞开始贴壁,细胞形态呈成纤维样,并很快形成集落。24h后首次换液,3天后细胞增殖加快,呈集落样迅速长,10～12天后细胞达到80%融合,3代后BMSCs呈梭形,细胞核较大、核仁明显,95%以上形态均匀一致,CD44及CD106相关抗原免疫荧光染色阳性,对照组未见荧光。同时向骨和脂肪组织诱导成功。
三、HUVECs、BMSCs在PHBHHx表面上的生长情况
在扫描电镜下可见,制备的材料为三维疏松多孔的结构。
与内皮细胞复合培养8、12、24h,可见材料表面均有细胞附着,细胞已伸展成梭形或不规则形,伸出伪足粘附材料上,细胞间无联系。在经过表面改性的材料表面,可见部分细胞跨越并覆盖孔隙,细胞连接增多。荧光显微镜下可见人脐静脉内皮细胞在加5%、10%中药涂层及碱处理的材料表面生长良好,已部分融合,且形态规则,具有铺路石样特征。然而在未经改性或未加药的材料表面较稀疏,分布不均匀。
与BMSCs复合培养1、3、7 d,可见材料表面均有细胞附着,细胞已伸展成梭形或多角形,伸出伪足粘附材料上,细胞表面有大量的微绒毛,并可见许多处于分裂相的细胞。中性红染色后观察,可见BMSCs在加5%、10%中药涂层或碱处理的材料表面生长良好,形态规则,且分布均匀,呈梭形或多边形,并有典型的集落样单位出现。然而在未经碱处理或未加中药涂层的材料表面细胞较稀疏,且形态不规则,无典型的集落样单位出现。
四、MTT法检测HUVECs、BMSCs在材料上的增殖情况
纵向比较,PHBHHx组和对照组的吸光度OD值都随时间延长而逐渐增高(p<0.05)。横向比较,0%未改性、0%改性、5%未改性、5%改性、10%未改性、10%改性各组的吸光度OD值都随组的顺序而逐渐升高(p<0.05),但对于HUVECs,0%改性和5%未改性,10%未改性和10%改性无显著性差异,对于BMSCs,0%改性组和5%未改性组无显著性差异。
结论
1、经表面改性处理的、且加入5%、10%中药涂层的PHBHHx膜,具有良好的人脐静脉内皮细胞相容性,在体外细胞培养的环境下,有利于细胞的生长、贴附和增殖。
2、经过碱处理及添加5%、10%中药涂层的PHBHHx材料与BMSCs有良好的体外相容性,可以作为一种新型材料而逐步应用于骨组织工程。
Objective
Atherosclerotic disease (AS) is a large class of diseases which harm seriously to human health, known as the "number one killer" in developed countries, and is one of the important reasons caused of death in the worldwide. Statistics show that the number of deaths worldwide in 1999 are about 58.6 million,in which the number of deaths due to coronary heart disease as high as 8.6 million, ranking No.1. China artery atherosclerotic disease incidence, although not as high as the western countries, but an upward trend. Mustardt said that the atherosclerotic heart disease is one of the causesd of morbidity and mortality, the disease makes the artery diameter stenosis and eventually blocks the blood vessel. In this context, vascular stents which has unique shape and biocompatibility are important technology for curing cardiovascular diseases, and has been widely used in China and abroad.
The ideal stent should have the following characteristics:(1) good biocompatibility, especially the blood compatibility, that is, non-induced thrombosis and non-immunogenicity; (2) appropriate mechanical properties, including appropriate intensity and compliance; (3) appropriate physical properties or chemical can make a better contraction/relaxation response of nerve stimulation; (4), short production cycle, low cost, and complete specifications and dimensions to meet clinical needs.
It is reported that 300 million people receive cardiovascular stent operations worldwide each year, but its implantation restenosis problem has never been able to be completely resolved. It conducted a multi-faceted efforts to try and found that the integrity of endothelial cells are damaged and dysfunction have a close relationship with restenosis after stent implantation, proposed to improve the endothelial cells (endothelial cells, EC) function and the integrity as a new strategy to prevent restenosis.
Endothelial cells is not only barrier between blood vessel wall and the blood, but also the body's active cells of metabolism, can synthesize and secrete a variety of bioactive substances, such as the secretion of nitrogen monoxidum, prostacyclin and other active substances, can be effective in preventing thrombus formation and maintenance of vascular contraction and relaxation, dynamic balance between blood clotting and anti-coagulation, form an anti-coagulation and anti-thrombotic system in the vascular cavity, and endothelial cells, blood cells have the same surface with a negative charge and thus has anti-platelet aggregation to prevent blood coagulation and thrombosis, thereby maintaining the normal blood flow and vascular patency long. The main difference between Artificial blood vessels and autologous vascular is no endothelial cells lining, so how to make artificial blood vessel endothelium is particularly important.
In summary, vascular stent endothelialization is one promising approach to resolve thrombosis and improve long-term patency rate after endovascular stent, through deeply study the surface/interface role among materials, specific cells,and tissue, and to study the materials science and biochemical factors, and the interaction between the two for cell adhesion, growth, proliferation, differentiation, gene expression and regulation, physiological functions and rules, then clarify the molecular recognition rules between biomedical materials and objectives substances, thereby strengthening the stent endothelialization research in vitro, to supplying possible for clinical application.
Polyhydroxy antibiotics (PHAs) are a class of copolymer,which can be naturally synthesized from many types of microorganisms, and have good biocompatibility and biodegradable. The first one in the family is PHAs, but the most in-depth research is a high crystallinity polymers,named 3-hydroxybutyric acid, PHB. Low molecular weight PHB,the final product in the blood is 3-hydroxybutyric acid copolyesters, a common metabolite, and in advanced animal experiments also proved that, PHB graft is non-toxic. However, PHB's brittleness and low mechanical strength significantly limits its wider application. Thus, PHA family, the materials has a better mechanical properties and thermodynamic stability, such as 3-hydroxybutyrate-co-3-hyd roxyhexanoate copolyesters (PHBHHx) will become a more practical value material. At present, the in vitro tests have demonstrated PHBHHx have very good cells biocompatibility, such as cartilage cells, bone mesenchymal stem cells and fibroblasts. By changing its composition, taking into account the different components can change the mechanical properties, I believe PHBHHx will be better performance to meet the demand of mechanical properties in bone tissue engineering and vascular tissue engineering. PHBHHx materials in this experiment by the NaOH treatment and adding different proportions of Chinese medicine-coating modification, using human umbilical vein endothelial cells, rat mesenchymal stem cells cultured to compound culture, then using morphology, immunocytochemistry, immunofluorescence cell chemistry and cell growth activity to explore the potential advantages in cardiovascular stent applications in the future, further promote the surface modified PHBHHx application early in the clinical medicine.
Methods
Male wistar rats weighing 100g were killed off cervical vertebra, through the whole bone marrow culture method to obtain relatively homogeneous BMSCs, cells seeded, fixed, for CD44, CD106 immunofluorescence staining identified. Obtained within 4 h post-natal health newborn umbilical cord, by trypsin digestion method to obtain a relatively homogeneous HUVECs, cells seeded, fixed, for vWF, FLK-1 immunochemical staining identified. Employing human umbilical vein endothelial cells, rat mesenchymal stem cells and surface modification of PHBHHx to co-culture, using scanning electron microscopy, immunofluorescence staining, neutral red staining and MTT method for analysising in vitro compatibility.
Results
1. HUVECs Isolation, culture and identification
3d after primary culture, HUVECs merge into single cells. After subculture, HUVECs is adherent in 30min,2-3d integration into a single layer. Morphological observation under inverted microscope, endothelial cells were spindle-shaped or polygonal, the size uniformity. After the formation of single-fusion cell growth, show a typical cobblestone or paving stone mosaic-like arrangement, and there is the phenomenon of contact inhibition, with endothelial cell characteristics. vWF, FLK-1 immunochemical staining can be seen noticeable brown granules within the cytoplasm, the nuclear-week intensive, whereas the control group no coloring.
2. BMSCs Isolation, culture and identification
BMSCs after primary culture 12h, the cells began to see a small number of adherent cells showed fibroblast-like morphology, and quickly form a colony, the first exchange of medium after 24h, after three days the cells speed up, showing the rapid colony-like grow, after 10 to 12 days the cells reached 80% confluence, three generations BMSCs spindle-shaped nucleus large, prominent nucleoli,95% or more patterns uniform. CD44 and CD106-associated antigen immunofluorescence staining position,but in the control group no fluorescence. At the same time be inducted to bone and fat tissue.
3. HUVECs, BMSCs growth condition in PHBHHx surface
In the scanning electron microscope, material apperent the three-dimensional porous structure.
And endothelial cell co-culture 8,12,24 h, can see cell attaching surface, the cell is being stretched into a spindle-shaped or irregular in shape, extending pseudopod adhesion material, cells without association. At surface modification materials surface, and seen part of cells across the pores, cell junction increasing. Visible under fluorescence microscopy,human umbilical vein endothelial cells in the plus 5%,10% of Chinese medicine coating and alkali-treated growth well, have been part of the integration, and with a paving stone-like characteristics. However, without modification or increase the drug surface scattered unevenly distributed.
BMSCs cocultured with 1,3,7 d, can be seen both cell attachment surface, the cell is being stretched into a spindle-shaped or polygonal, extending pseudopodia binding material on a large number of cell surface microvilli, and shows that many are split-phase cells. Observed after neutral red staining, showing that BMSCs at plus 5%, 10% of Chinese medicine coating or surface treatment grew well, morphological rules, and distributed evenly, showing spindle-shaped or polygonal and have the typical colony like the flats. However, without alkali treatment or traditional Chinese medicine for coating the surface of cells, the material is light, and the irregular shape, non-typical colony like the flats
4.MTT assay HUVECs, BMSCs proliferation condition on the material surface
Vertical comparison, PHBHHx group and the control group OD value with time was gradually increased (p<0.05). Horizontal comparison,0% non-modified,0% modified, 5% non-modified,5% modified,10% unmodified,10% modified the absorbance OD values of each group with the group in the order are gradually increased (p<0.05), but for HUVECs,0% modified and 5% non-modified,10% and 10% of the unmodified modification was no significant difference, for BMSCs,0% modified group and 5% improved for There were no significant differences.
Conclusions
1. Through surface modification treatment, and the addition of 5%,10% of Chinese medicine coating PHBHHx film has a good human umbilical vein endothelial cell compatibility,which is conducive to cell growth, pasted and proliferation in vitro cell culture environment.
2. After alkali treatment and adding 5%,10% of Chinese medicine PHBHHx coating materials and BMSCs in vitro have good compatibility, can be used as a new type of materials used in bone tissue engineering phase.
动脉粥样硬化性疾病(AS)是严重危害人类健康的一大类疾病,在发达国家被称为“头号杀手”,是世界范围的主要死亡原因之一。资料显示,1999年全世界死亡人数约5860万.其中因冠心病死亡人数高达860万,位居死因第1位。我国动脉粥样硬化性疾病发病虽没有西方国家那么高,但正呈上升趋势发展。Mustardt等指出,动脉粥样硬化心脏病是导致发病和死亡的原因之一,这种疾病使得动脉管径狭窄,最终使血管堵塞。在此背景下,血管支架因其独特的成型性和生物相容性成为治疗心血管疾病的重要技术,近年来在国内外已被广泛应用。
理想的血管支架应具有以下特征：(1)良好的生物相容性,尤其是血液相容性,即非致血栓性和无免疫原性；(2)适当的机械性能,包括适当的强度和顺应性；(3)适当的生理性能,对神经或化学刺激能做出较好收缩／舒张反应；(4)生产周期短、成本低,而且规格和尺寸齐全能满足临床需要。
据报道,全世界每年有300万人接受心血管支架置人手术,但其置入术后血管再狭窄问题却始终未能得到彻底解决。人们进行了多方面的尝试和努力,发现内皮细胞完整性的破坏及功能紊乱与支架植入后血管再狭窄有着密切的关系,提出将改善内皮细胞(endothelial cells, EC)功能及完整性的恢复作为预防再狭窄的新策略。
内皮细胞不仅是介于血管壁和血液之间的屏障结构。而且是体内一种代谢十分活跃的细胞,能合成和分泌多种生物活性物质,如分泌一氧化氮、前列环素等活性物质,可有效防止血栓形成,维持血管收缩、舒张,凝血与抗凝血等动态平衡,在血管腔的表面形成一抗凝血和抗血栓系统,另外内皮细胞同血细胞一样表面带负电荷,因而具有抗血小板聚集、防止血液凝固和血栓形成的作用,从而保持血液的正常流动和血管的长通畅。人工血管与自体血管的主要区别是无内皮细胞衬里,所以如何使人工血管内皮化,显得尤为重要。
总之,血管内支架内皮化是解决血管内支架置人后血栓形成和提高远期通畅率很有前途的方法之一,通过从细胞和分子水平深入研究材料与特定细胞、组织之间的表面／界面作用,并研究材料学因素和生物化学因素及二者相互作用对细胞的黏附、生长、增殖、分化、基因表达和调控、生理功能的影响因素和规律,进而阐明生物医学材料与目标物质之间的分子识别规律,进而加强血管内支架体外内皮化的基础研究将为其临床应用效果的改善成为可能。
多羟基抗生素类(PHAs)是一类可以由很多种类微生物自然产生的,具有良好生物相容性的生物可降解的共聚物。在PHAs家族中第一个,也是研究最深入的就是具有高结晶性的3-羟基丁酸聚合物,即PHB。低分子量的PHB在血液中的终产物是3-羟基丁酸,一种普通的代谢产物,并且在高级动物的试验中也证明,PHB移植物是无毒的。然而,PHB的脆性和低力学强度明显限制了它的广泛应用。因此,PHA家族中具有更好机械学性能和热力学稳定性的材料,诸如3-羟基丁酸与3-羟基己酸共聚酯(PHBHHx)便成为了更有实际应用价值的材料。目前,体外试验已证明了PHBHHx对一些细胞的生物相容性非常好,如软骨细胞,骨髓间充质干细胞和成纤维细胞。考虑到通过改变其组成的不同组分可以改变它的力学特性的特点,相信PHBHHx一定能更好的满足力学特性要求高的骨组织工程和弹性要求好的血管组织工程的要求。本实验PHBHHx材料经NaOH处理及添加了不同比例的中药涂层改性后,采用人脐静脉内皮细胞、大鼠间充质干细胞与其进行复合培养后,利用形态学、免疫细胞化学、免疫荧光细胞化学以及细胞生长活力等多个指标进行分析,从而探讨表面改性PHBHHx在将来心血管支架应用中的潜在优势,进一步促进经过表面改性的PHBHHx在临床的早日应用。
方法
体重100g雄性wistar大鼠脱颈椎处死,通过全骨髓培养法获得相对均质的BMSCs,细胞爬片,固定,进行CD44、CD 106免疫荧光染色鉴定。取产后4h内的健康新生儿脐带,通过胰酶消化法,获得相对均质的HUVECs,细胞爬片,固定,进行vWF、FLK-1免疫化学染色鉴定。用人脐静脉内皮细胞、大鼠间充质干细胞与表面改性的PHBHHx进行复合培养后,利用扫描电镜观察、免疫荧光染色、中性红染色及MTT法等多个指标进行分析其体外相容性。
结果
一、HUVECs的分离、培养及鉴定
原代培养3d后,HUVECs汇合成单层细胞。传代培养的HUVECs 30min即贴壁,2-3d融合成单层。形态学观察倒置显微镜下内皮细胞呈梭形或多边形,大小均匀。细胞生长融合形成单层后,呈典型的鹅卵石或铺路石镶嵌状排列生长,并有接触抑制现象,具有内皮细胞特征。vWF、FLK-1免疫化学染色显可见胞质内棕黄色颗粒,核周密集,而对照组内未见着色。
二、MSCs的分离、培养及鉴定
BMSCs原代培养接种后12h可见少数细胞开始贴壁,细胞形态呈成纤维样,并很快形成集落。24h后首次换液,3天后细胞增殖加快,呈集落样迅速长,10～12天后细胞达到80%融合,3代后BMSCs呈梭形,细胞核较大、核仁明显,95%以上形态均匀一致,CD44及CD106相关抗原免疫荧光染色阳性,对照组未见荧光。同时向骨和脂肪组织诱导成功。
三、HUVECs、BMSCs在PHBHHx表面上的生长情况
在扫描电镜下可见,制备的材料为三维疏松多孔的结构。
与内皮细胞复合培养8、12、24h,可见材料表面均有细胞附着,细胞已伸展成梭形或不规则形,伸出伪足粘附材料上,细胞间无联系。在经过表面改性的材料表面,可见部分细胞跨越并覆盖孔隙,细胞连接增多。荧光显微镜下可见人脐静脉内皮细胞在加5%、10%中药涂层及碱处理的材料表面生长良好,已部分融合,且形态规则,具有铺路石样特征。然而在未经改性或未加药的材料表面较稀疏,分布不均匀。
与BMSCs复合培养1、3、7 d,可见材料表面均有细胞附着,细胞已伸展成梭形或多角形,伸出伪足粘附材料上,细胞表面有大量的微绒毛,并可见许多处于分裂相的细胞。中性红染色后观察,可见BMSCs在加5%、10%中药涂层或碱处理的材料表面生长良好,形态规则,且分布均匀,呈梭形或多边形,并有典型的集落样单位出现。然而在未经碱处理或未加中药涂层的材料表面细胞较稀疏,且形态不规则,无典型的集落样单位出现。
四、MTT法检测HUVECs、BMSCs在材料上的增殖情况
纵向比较,PHBHHx组和对照组的吸光度OD值都随时间延长而逐渐增高(p<0.05)。横向比较,0%未改性、0%改性、5%未改性、5%改性、10%未改性、10%改性各组的吸光度OD值都随组的顺序而逐渐升高(p<0.05),但对于HUVECs,0%改性和5%未改性,10%未改性和10%改性无显著性差异,对于BMSCs,0%改性组和5%未改性组无显著性差异。
结论
1、经表面改性处理的、且加入5%、10%中药涂层的PHBHHx膜,具有良好的人脐静脉内皮细胞相容性,在体外细胞培养的环境下,有利于细胞的生长、贴附和增殖。
2、经过碱处理及添加5%、10%中药涂层的PHBHHx材料与BMSCs有良好的体外相容性,可以作为一种新型材料而逐步应用于骨组织工程。
Objective
Atherosclerotic disease (AS) is a large class of diseases which harm seriously to human health, known as the "number one killer" in developed countries, and is one of the important reasons caused of death in the worldwide. Statistics show that the number of deaths worldwide in 1999 are about 58.6 million,in which the number of deaths due to coronary heart disease as high as 8.6 million, ranking No.1. China artery atherosclerotic disease incidence, although not as high as the western countries, but an upward trend. Mustardt said that the atherosclerotic heart disease is one of the causesd of morbidity and mortality, the disease makes the artery diameter stenosis and eventually blocks the blood vessel. In this context, vascular stents which has unique shape and biocompatibility are important technology for curing cardiovascular diseases, and has been widely used in China and abroad.
The ideal stent should have the following characteristics:(1) good biocompatibility, especially the blood compatibility, that is, non-induced thrombosis and non-immunogenicity; (2) appropriate mechanical properties, including appropriate intensity and compliance; (3) appropriate physical properties or chemical can make a better contraction/relaxation response of nerve stimulation; (4), short production cycle, low cost, and complete specifications and dimensions to meet clinical needs.
It is reported that 300 million people receive cardiovascular stent operations worldwide each year, but its implantation restenosis problem has never been able to be completely resolved. It conducted a multi-faceted efforts to try and found that the integrity of endothelial cells are damaged and dysfunction have a close relationship with restenosis after stent implantation, proposed to improve the endothelial cells (endothelial cells, EC) function and the integrity as a new strategy to prevent restenosis.
Endothelial cells is not only barrier between blood vessel wall and the blood, but also the body's active cells of metabolism, can synthesize and secrete a variety of bioactive substances, such as the secretion of nitrogen monoxidum, prostacyclin and other active substances, can be effective in preventing thrombus formation and maintenance of vascular contraction and relaxation, dynamic balance between blood clotting and anti-coagulation, form an anti-coagulation and anti-thrombotic system in the vascular cavity, and endothelial cells, blood cells have the same surface with a negative charge and thus has anti-platelet aggregation to prevent blood coagulation and thrombosis, thereby maintaining the normal blood flow and vascular patency long. The main difference between Artificial blood vessels and autologous vascular is no endothelial cells lining, so how to make artificial blood vessel endothelium is particularly important.
In summary, vascular stent endothelialization is one promising approach to resolve thrombosis and improve long-term patency rate after endovascular stent, through deeply study the surface/interface role among materials, specific cells,and tissue, and to study the materials science and biochemical factors, and the interaction between the two for cell adhesion, growth, proliferation, differentiation, gene expression and regulation, physiological functions and rules, then clarify the molecular recognition rules between biomedical materials and objectives substances, thereby strengthening the stent endothelialization research in vitro, to supplying possible for clinical application.
Polyhydroxy antibiotics (PHAs) are a class of copolymer,which can be naturally synthesized from many types of microorganisms, and have good biocompatibility and biodegradable. The first one in the family is PHAs, but the most in-depth research is a high crystallinity polymers,named 3-hydroxybutyric acid, PHB. Low molecular weight PHB,the final product in the blood is 3-hydroxybutyric acid copolyesters, a common metabolite, and in advanced animal experiments also proved that, PHB graft is non-toxic. However, PHB's brittleness and low mechanical strength significantly limits its wider application. Thus, PHA family, the materials has a better mechanical properties and thermodynamic stability, such as 3-hydroxybutyrate-co-3-hyd roxyhexanoate copolyesters (PHBHHx) will become a more practical value material. At present, the in vitro tests have demonstrated PHBHHx have very good cells biocompatibility, such as cartilage cells, bone mesenchymal stem cells and fibroblasts. By changing its composition, taking into account the different components can change the mechanical properties, I believe PHBHHx will be better performance to meet the demand of mechanical properties in bone tissue engineering and vascular tissue engineering. PHBHHx materials in this experiment by the NaOH treatment and adding different proportions of Chinese medicine-coating modification, using human umbilical vein endothelial cells, rat mesenchymal stem cells cultured to compound culture, then using morphology, immunocytochemistry, immunofluorescence cell chemistry and cell growth activity to explore the potential advantages in cardiovascular stent applications in the future, further promote the surface modified PHBHHx application early in the clinical medicine.
Methods
Male wistar rats weighing 100g were killed off cervical vertebra, through the whole bone marrow culture method to obtain relatively homogeneous BMSCs, cells seeded, fixed, for CD44, CD106 immunofluorescence staining identified. Obtained within 4 h post-natal health newborn umbilical cord, by trypsin digestion method to obtain a relatively homogeneous HUVECs, cells seeded, fixed, for vWF, FLK-1 immunochemical staining identified. Employing human umbilical vein endothelial cells, rat mesenchymal stem cells and surface modification of PHBHHx to co-culture, using scanning electron microscopy, immunofluorescence staining, neutral red staining and MTT method for analysising in vitro compatibility.
Results
1. HUVECs Isolation, culture and identification
3d after primary culture, HUVECs merge into single cells. After subculture, HUVECs is adherent in 30min,2-3d integration into a single layer. Morphological observation under inverted microscope, endothelial cells were spindle-shaped or polygonal, the size uniformity. After the formation of single-fusion cell growth, show a typical cobblestone or paving stone mosaic-like arrangement, and there is the phenomenon of contact inhibition, with endothelial cell characteristics. vWF, FLK-1 immunochemical staining can be seen noticeable brown granules within the cytoplasm, the nuclear-week intensive, whereas the control group no coloring.
2. BMSCs Isolation, culture and identification
BMSCs after primary culture 12h, the cells began to see a small number of adherent cells showed fibroblast-like morphology, and quickly form a colony, the first exchange of medium after 24h, after three days the cells speed up, showing the rapid colony-like grow, after 10 to 12 days the cells reached 80% confluence, three generations BMSCs spindle-shaped nucleus large, prominent nucleoli,95% or more patterns uniform. CD44 and CD106-associated antigen immunofluorescence staining position,but in the control group no fluorescence. At the same time be inducted to bone and fat tissue.
3. HUVECs, BMSCs growth condition in PHBHHx surface
In the scanning electron microscope, material apperent the three-dimensional porous structure.
And endothelial cell co-culture 8,12,24 h, can see cell attaching surface, the cell is being stretched into a spindle-shaped or irregular in shape, extending pseudopod adhesion material, cells without association. At surface modification materials surface, and seen part of cells across the pores, cell junction increasing. Visible under fluorescence microscopy,human umbilical vein endothelial cells in the plus 5%,10% of Chinese medicine coating and alkali-treated growth well, have been part of the integration, and with a paving stone-like characteristics. However, without modification or increase the drug surface scattered unevenly distributed.
BMSCs cocultured with 1,3,7 d, can be seen both cell attachment surface, the cell is being stretched into a spindle-shaped or polygonal, extending pseudopodia binding material on a large number of cell surface microvilli, and shows that many are split-phase cells. Observed after neutral red staining, showing that BMSCs at plus 5%, 10% of Chinese medicine coating or surface treatment grew well, morphological rules, and distributed evenly, showing spindle-shaped or polygonal and have the typical colony like the flats. However, without alkali treatment or traditional Chinese medicine for coating the surface of cells, the material is light, and the irregular shape, non-typical colony like the flats
4.MTT assay HUVECs, BMSCs proliferation condition on the material surface
Vertical comparison, PHBHHx group and the control group OD value with time was gradually increased (p<0.05). Horizontal comparison,0% non-modified,0% modified, 5% non-modified,5% modified,10% unmodified,10% modified the absorbance OD values of each group with the group in the order are gradually increased (p<0.05), but for HUVECs,0% modified and 5% non-modified,10% and 10% of the unmodified modification was no significant difference, for BMSCs,0% modified group and 5% improved for There were no significant differences.
Conclusions
1. Through surface modification treatment, and the addition of 5%,10% of Chinese medicine coating PHBHHx film has a good human umbilical vein endothelial cell compatibility,which is conducive to cell growth, pasted and proliferation in vitro cell culture environment.
2. After alkali treatment and adding 5%,10% of Chinese medicine PHBHHx coating materials and BMSCs in vitro have good compatibility, can be used as a new type of materials used in bone tissue engineering phase.
引文
1 Gnecchi M, Melo LG. Bone marrow-derived mesenchymal stem cells:isolation, expansion, characterization,viral transduction, and production of conditioned medium. Methods Mol Biol.2009;482:281-94.
2 Haiying Yu, Paul H Wooley. et al. Biocompatibility of Poly-ε-caprolactone-hydroxyapatite composite on mouse bone marrow-derived osteoblasts and endothelial cells. J orthop Surg Re.2009;26(4)5.
3 Champa A, Ebraheim NA.Evaluation of bone matrix and demineralized bone matrix incorporated PLGA matrices for bone repair. J Mater Sci Mater Med.2009;Mar 29. [Epub ahead of print]
4 Esposito MT, Di Noto R, et al. Culture Conditions Allow Selection of Different Mesenchymal Progenitors from Adult Mouse Bone Marrow. Tissue Eng Part A. 2009;Mar 17. [Epub ahead of print]
5 Wu Q, Wang Y, et al. Medical application of microbial biopolyesters polyhydroxyalkanoates. Artif Cells Blood Substit Immobil Biotechnol.2009;37(1):1-12.
6 Eble JA, Niland S.The extracellular matrix of blood vessels. Curr Pharm Des. 2009;15(12):1385-400.
7 Krenning G, Moonen JR. et al. Generating new blood flow:integrating developmental biology and tissue engineering.Trends Cardiovasc Med.2008; Nov;18(8):312-23.
8 Uchida T, Ikeda S, et al. Development of biodegradable scaffolds based on patient-specific arterial configuration.. J Biotechnol.2008;Jan 20;133(2):213-8. Epub 2007 Aug 14.
9 Jing Xi,Ling Zhang, et al. Preparation and evaluation of porous poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) hydroxyapatite composite scaffolds. J Biomater Appl,2008;22(4):293-307.
10 McLucas E, Rochev Y,et al. Analysis of the effects of surface treatments on nickel release from nitinol wires and their impact on candidate gene expression in endothelial cells. J Mater Sci Mater Med.2008; Mar;19(3):975-80. Epub 2008 Feb 5.
11 Taylor SE, Smith RK, et al. Mesenchymal stem cell therapy in equine musculoskeletal disease:scientific fact or clinical fiction? Equine Vet J.2007; Mar,39(2):172-80.
12 Prasad CK, Krishnan LK. Regulation of endothelial cell phenotype by biomimetic matrix coated on biomaterials for cardiovascular tissue engineering. Acta Biomater. 2008;Jan;4(1):182-91. Epub 2007 Jul 3.
13 Baudin B, Bruneel A, et al. A protocol for isolation and culture of human umbilical vein endothelial cells.Nat Protoc.2007;2(3):481-5.
14 Vilquin JT,Rosset P. Mesenchymal stem cells in bone and cartilage repair:current
status. Regen Med.2006; Jul.1(4):589-604.
15 Yang M, Zhu S, et al. Studies on bone marrow stromal cells affinity of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate). Biomaterials. 2004;Mar-Apr;25(7-8):1365-73.
16 Boura C, Menu P, et al. Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification..Biomaterials.2003;Sep;24(20):35 21-30.
17 Tiwari A,Salacinski HJ.Hamilton G, et al.Tissue engineering of vascular bypass grafts: role of endothelial cell extraction..Eur J Vasc Endovasc Surg:2001;21:193-201.
18 Kirkpatrick CJ, Otto M, et al. Endothelial cell cultures as a tool in biomaterial research.. J Mater Sci Mater Med.1999; Oct-Nov;10(10/11):589-94.
19 Herring M,Gardner A, et al. A Single-staged technique for seeding vascular grafts with autogenous endothelium..Surgery.1978;84:498-504.
20王宏天,马继伟.阿魏酸钠对缺血性急性肾衰竭中SDF-1表达影响.中国中西医结合肾病杂志.2009；10(2)：113-115.
21 王健,张海燕,胡勇等.骨髓间充质干细胞复合羟基磷灰石／磷酸三钙植骨材料的体外研究..解剖学报.2008；39(4)：539-542.
22王慧君,柏树令.毛囊培养上清液诱导大鼠骨髓间充质干细胞分化的研究.解剖学报.2008；39(3)：386-89.
23 陈华勇,柏树令,王军等.异种小血管支架模型的建立.中国修复重建外科杂志.2005；Vol 19(5)：392-394.
24潘锋,柏树令.兔BMSCs体外培养及其向成骨细胞分化的实验研究.解剖科学进展.2005；11(1)：12-15.
1 Yang F. Williams CG. et al.The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells.Biomaterials.2005;26(30):5991-5998.
2 Anselme K, Bigerelle M. Topography effects of pure titanium substrates on human osteoblast long-term adhesion.Acta Biomater.2005;1(2):211-222
3 Lim JY, Dreiss AD. et al.The regulation of integrin-mediated osteoblast focal adhesion and focal adhesion kinase expression by nanoscale topography.Biomaterials.2007;28(10): 1787-1797.
4 Lim JY, Hansen JC, SiedleckiCA.et al. et al. Human foetal osteoblastic cell response to polymer-demixed nanotopographic interfaces.JR Soc Interface,2005.2(2);97-108
5 Nugent H M, Edelman E R. Tissue engineering therapy for cardiovascular disease. Circ Res.2003;92:1068—1078.
6 Anselme K. Osteoblast adhesion on biomaterials. Biomaterials.2000;21:667.681.
7 Puleo DA, Nanci A. Understanding and controlling thebone-implant interface. Biomaterials.1999;20:2311-2321
8 蓝旭,梁军,葛宝丰,刘雪梅.表面修饰对羟基磷灰石修复骨缺损的影响.生物医学工程与临床.2008;vol.12,No.2
9 黄岳山,赵修华,吴源青.微弧氧化改性二氧化钛生物活性膜的研究进展.医疗卫生装备.2004;4：22～23.
10战德松,杨彦昌.钛表面涂敷类金刚石碳膜的生物相容性研究.材料研究学报.1999;13：405-408
11 BagnoA, Piovan A, et al. Human osteoblast-like cell adhesion on titanium substrates covalently functionalized with synthetic peptides.Bone.2007;40(3):693-699
12 GuoKT, ScharnweberD, et al. The effect of electrochemical functionalization of Ti-alloy surfaces by aptamer-based capture molecules on cell adhesion.Biomaterials.2007;28(3): 468-474.
13 RamiFes PA, Mirer IL, et al.Hasma-treated PET surfaces improvethe biocompatibility of human endothelial cells. J Biomed Mater Res.2000;51(3):535-539.
14 Pislaru SV, Harbuzariu A, et al. Magnetic forces enable rapid endothelialization of synthetic vascular grafts. Circulation.2006;114(1-suppl):1314-1318.
15 Bacakova L, Mares V, et al. Molecular mechanisms of Improved adhesion and growth of an endothelial cell linecultured on polystyrene implanted with fluorine ions. Biomaterials.2000;21 (11):1173-1179.
16 Bowlin GL, Rittgers SE. In vitro evaluation of electmstatic endothelial cell transplantion
onto 4 mm interior diameter expanded polytetrafluoroetthylene grafts. J Vasc Surg.1998;27(3):504.
17 Rosenman JE. Kempezinski RE et al.Kinetics of Endothdial cell seeding. J Vasc Surg.1995;2(6):778-784.
18 Massia SP. Hubhell JA. Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials. J Biomed Mater Res.1993;25(13):223-242.
19 Hsu SH, Sun SH, et al. Improved retention of endothelial cell seeded on polyurethane small-diameter vascular grafts modified by a recombinant RGD-containing protein. Artif Organs.2003;27(12):1068-1078.
20 Li C, Hill A, Incan M. In vitro and in vivo studies of ePTFE vascular graft treated with P15 poptide. J Biomater Sci Polym Ed.2005;16(7):875-891.
21 Zarge JI, Husak V, et al. Fibrin glue containing fibreblast growth factor type I and heparin decreases platelet deposition. Am J Sure.1997; 174(2):188-192.
22 Crombez M, Chevallier P, et al. Improving arterial presthesis neo-endothelialization: application of a proactive VEGF construct onto PTEE surfaces. Biomaterials.2005;26(35): 7402-7409.
23 Randone B, Cavallaro G, et al. Dual role of VEGF in pretreated experimental ePTFE arterial graft. J Surg Res.2005; 127(2):70-79.
24 Sudesh K, Abe H, et al. Synthesis, structure and properties of poly-hydroxyalkanoates: biological polyesters. Prog Polym Sci.2000(17);25:1503-55.
25 Williams SF, Martin DP. Application of PHAs in medicine and pharmacy. In:Doi Y, Stenbu chel A, editors. Biopolymers (Polyester Ⅲ).2002(7);26.91-128.
26 Saito T, Tomita K, Juni K, et al. In vivo and in vitro degradation of poly(3-hydroxybutyrate) in rat. Biomaterials.1991;12:309-12.
27 Gogolewski S, Jovanovic M, et al. Tissue response and in vivo degradation of selected polyhydroxyacids:polylactides (PLA), poly(3-hydroxybutyrate) (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/VA). J Biomed Mater Res.1993;27:1135-48.
28 Ya-Wu Wang, Qiong Wu*, et al. Attachment, proliferation and differentiation of osteoblasts on random biopolyester poly(3-hydroxybutyrate -co-3-hydroxyhexanoate) scaffolds. Biomaterials.2004;28(2):669-675.
29 Reusch RN, Sparrow AJ, et al. Transport of poly betahydroxybutyrate in human plasma. Biochim Biophys Acta.1992; 1123:33-40.
30 Yang XS, Zhao K, et al. Effect of surface treatment on the biocompatibility of microbial polyhydroxyalkanoates. Biomaterials.2002;23(5):1391-7.
31 Zhao K. Yang X. et al. Effect of lipase treatment on the biocompatibility of microbial polyhydroxyalkanoates. J Mater Sci-Mater Med.2002;13(9):849-54.
32 Zheng Z, Bei FF. et al. Effects of crystallization of polyhydroxyalkanoate blend on surface physicochemical properties and interactions with rabbit articular cartilage chondrocytes.Biomaterials.2005(2);26:3537-48.
33 Wang YW. Mo WK, et al. Biodegradation studies of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Polym Degrad Stability.2004(2);85:815-22.
34 Zhao K, Deng Y, et al. Polyhydroxyalkanoate (PHA) scaffolds with good mechanical properties and biocompatibility.Biomaterials.2003;24(13):1041-5.
35 Wang YW, Wu Q, et al. Reduced mouse cell growth by increased hydrophilicity of microbial polyhydroxyalkanoates via hyaluronan coating. Biomaterials.2005(7);24:4621-9.
36 Yang M, Zhu SS et al. Studies on bone marrow stromal cells affinity of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate). Biomaterials.2006;25(9):1365-73.
2 Haiying Yu, Paul H Wooley. et al. Biocompatibility of Poly-ε-caprolactone-hydroxyapatite composite on mouse bone marrow-derived osteoblasts and endothelial cells. J orthop Surg Re.2009;26(4)5.
3 Champa A, Ebraheim NA.Evaluation of bone matrix and demineralized bone matrix incorporated PLGA matrices for bone repair. J Mater Sci Mater Med.2009;Mar 29. [Epub ahead of print]
4 Esposito MT, Di Noto R, et al. Culture Conditions Allow Selection of Different Mesenchymal Progenitors from Adult Mouse Bone Marrow. Tissue Eng Part A. 2009;Mar 17. [Epub ahead of print]
5 Wu Q, Wang Y, et al. Medical application of microbial biopolyesters polyhydroxyalkanoates. Artif Cells Blood Substit Immobil Biotechnol.2009;37(1):1-12.
6 Eble JA, Niland S.The extracellular matrix of blood vessels. Curr Pharm Des. 2009;15(12):1385-400.
7 Krenning G, Moonen JR. et al. Generating new blood flow:integrating developmental biology and tissue engineering.Trends Cardiovasc Med.2008; Nov;18(8):312-23.
8 Uchida T, Ikeda S, et al. Development of biodegradable scaffolds based on patient-specific arterial configuration.. J Biotechnol.2008;Jan 20;133(2):213-8. Epub 2007 Aug 14.
9 Jing Xi,Ling Zhang, et al. Preparation and evaluation of porous poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) hydroxyapatite composite scaffolds. J Biomater Appl,2008;22(4):293-307.
10 McLucas E, Rochev Y,et al. Analysis of the effects of surface treatments on nickel release from nitinol wires and their impact on candidate gene expression in endothelial cells. J Mater Sci Mater Med.2008; Mar;19(3):975-80. Epub 2008 Feb 5.
11 Taylor SE, Smith RK, et al. Mesenchymal stem cell therapy in equine musculoskeletal disease:scientific fact or clinical fiction? Equine Vet J.2007; Mar,39(2):172-80.
12 Prasad CK, Krishnan LK. Regulation of endothelial cell phenotype by biomimetic matrix coated on biomaterials for cardiovascular tissue engineering. Acta Biomater. 2008;Jan;4(1):182-91. Epub 2007 Jul 3.
13 Baudin B, Bruneel A, et al. A protocol for isolation and culture of human umbilical vein endothelial cells.Nat Protoc.2007;2(3):481-5.
14 Vilquin JT,Rosset P. Mesenchymal stem cells in bone and cartilage repair:current
status. Regen Med.2006; Jul.1(4):589-604.
15 Yang M, Zhu S, et al. Studies on bone marrow stromal cells affinity of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate). Biomaterials. 2004;Mar-Apr;25(7-8):1365-73.
16 Boura C, Menu P, et al. Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification..Biomaterials.2003;Sep;24(20):35 21-30.
17 Tiwari A,Salacinski HJ.Hamilton G, et al.Tissue engineering of vascular bypass grafts: role of endothelial cell extraction..Eur J Vasc Endovasc Surg:2001;21:193-201.
18 Kirkpatrick CJ, Otto M, et al. Endothelial cell cultures as a tool in biomaterial research.. J Mater Sci Mater Med.1999; Oct-Nov;10(10/11):589-94.
19 Herring M,Gardner A, et al. A Single-staged technique for seeding vascular grafts with autogenous endothelium..Surgery.1978;84:498-504.
20王宏天,马继伟.阿魏酸钠对缺血性急性肾衰竭中SDF-1表达影响.中国中西医结合肾病杂志.2009；10(2)：113-115.
21 王健,张海燕,胡勇等.骨髓间充质干细胞复合羟基磷灰石／磷酸三钙植骨材料的体外研究..解剖学报.2008；39(4)：539-542.
22王慧君,柏树令.毛囊培养上清液诱导大鼠骨髓间充质干细胞分化的研究.解剖学报.2008；39(3)：386-89.
23 陈华勇,柏树令,王军等.异种小血管支架模型的建立.中国修复重建外科杂志.2005；Vol 19(5)：392-394.
24潘锋,柏树令.兔BMSCs体外培养及其向成骨细胞分化的实验研究.解剖科学进展.2005；11(1)：12-15.
1 Yang F. Williams CG. et al.The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells.Biomaterials.2005;26(30):5991-5998.
2 Anselme K, Bigerelle M. Topography effects of pure titanium substrates on human osteoblast long-term adhesion.Acta Biomater.2005;1(2):211-222
3 Lim JY, Dreiss AD. et al.The regulation of integrin-mediated osteoblast focal adhesion and focal adhesion kinase expression by nanoscale topography.Biomaterials.2007;28(10): 1787-1797.
4 Lim JY, Hansen JC, SiedleckiCA.et al. et al. Human foetal osteoblastic cell response to polymer-demixed nanotopographic interfaces.JR Soc Interface,2005.2(2);97-108
5 Nugent H M, Edelman E R. Tissue engineering therapy for cardiovascular disease. Circ Res.2003;92:1068—1078.
6 Anselme K. Osteoblast adhesion on biomaterials. Biomaterials.2000;21:667.681.
7 Puleo DA, Nanci A. Understanding and controlling thebone-implant interface. Biomaterials.1999;20:2311-2321
8 蓝旭,梁军,葛宝丰,刘雪梅.表面修饰对羟基磷灰石修复骨缺损的影响.生物医学工程与临床.2008;vol.12,No.2
9 黄岳山,赵修华,吴源青.微弧氧化改性二氧化钛生物活性膜的研究进展.医疗卫生装备.2004;4：22～23.
10战德松,杨彦昌.钛表面涂敷类金刚石碳膜的生物相容性研究.材料研究学报.1999;13：405-408
11 BagnoA, Piovan A, et al. Human osteoblast-like cell adhesion on titanium substrates covalently functionalized with synthetic peptides.Bone.2007;40(3):693-699
12 GuoKT, ScharnweberD, et al. The effect of electrochemical functionalization of Ti-alloy surfaces by aptamer-based capture molecules on cell adhesion.Biomaterials.2007;28(3): 468-474.
13 RamiFes PA, Mirer IL, et al.Hasma-treated PET surfaces improvethe biocompatibility of human endothelial cells. J Biomed Mater Res.2000;51(3):535-539.
14 Pislaru SV, Harbuzariu A, et al. Magnetic forces enable rapid endothelialization of synthetic vascular grafts. Circulation.2006;114(1-suppl):1314-1318.
15 Bacakova L, Mares V, et al. Molecular mechanisms of Improved adhesion and growth of an endothelial cell linecultured on polystyrene implanted with fluorine ions. Biomaterials.2000;21 (11):1173-1179.
16 Bowlin GL, Rittgers SE. In vitro evaluation of electmstatic endothelial cell transplantion
onto 4 mm interior diameter expanded polytetrafluoroetthylene grafts. J Vasc Surg.1998;27(3):504.
17 Rosenman JE. Kempezinski RE et al.Kinetics of Endothdial cell seeding. J Vasc Surg.1995;2(6):778-784.
18 Massia SP. Hubhell JA. Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials. J Biomed Mater Res.1993;25(13):223-242.
19 Hsu SH, Sun SH, et al. Improved retention of endothelial cell seeded on polyurethane small-diameter vascular grafts modified by a recombinant RGD-containing protein. Artif Organs.2003;27(12):1068-1078.
20 Li C, Hill A, Incan M. In vitro and in vivo studies of ePTFE vascular graft treated with P15 poptide. J Biomater Sci Polym Ed.2005;16(7):875-891.
21 Zarge JI, Husak V, et al. Fibrin glue containing fibreblast growth factor type I and heparin decreases platelet deposition. Am J Sure.1997; 174(2):188-192.
22 Crombez M, Chevallier P, et al. Improving arterial presthesis neo-endothelialization: application of a proactive VEGF construct onto PTEE surfaces. Biomaterials.2005;26(35): 7402-7409.
23 Randone B, Cavallaro G, et al. Dual role of VEGF in pretreated experimental ePTFE arterial graft. J Surg Res.2005; 127(2):70-79.
24 Sudesh K, Abe H, et al. Synthesis, structure and properties of poly-hydroxyalkanoates: biological polyesters. Prog Polym Sci.2000(17);25:1503-55.
25 Williams SF, Martin DP. Application of PHAs in medicine and pharmacy. In:Doi Y, Stenbu chel A, editors. Biopolymers (Polyester Ⅲ).2002(7);26.91-128.
26 Saito T, Tomita K, Juni K, et al. In vivo and in vitro degradation of poly(3-hydroxybutyrate) in rat. Biomaterials.1991;12:309-12.
27 Gogolewski S, Jovanovic M, et al. Tissue response and in vivo degradation of selected polyhydroxyacids:polylactides (PLA), poly(3-hydroxybutyrate) (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/VA). J Biomed Mater Res.1993;27:1135-48.
28 Ya-Wu Wang, Qiong Wu*, et al. Attachment, proliferation and differentiation of osteoblasts on random biopolyester poly(3-hydroxybutyrate -co-3-hydroxyhexanoate) scaffolds. Biomaterials.2004;28(2):669-675.
29 Reusch RN, Sparrow AJ, et al. Transport of poly betahydroxybutyrate in human plasma. Biochim Biophys Acta.1992; 1123:33-40.
30 Yang XS, Zhao K, et al. Effect of surface treatment on the biocompatibility of microbial polyhydroxyalkanoates. Biomaterials.2002;23(5):1391-7.
31 Zhao K. Yang X. et al. Effect of lipase treatment on the biocompatibility of microbial polyhydroxyalkanoates. J Mater Sci-Mater Med.2002;13(9):849-54.
32 Zheng Z, Bei FF. et al. Effects of crystallization of polyhydroxyalkanoate blend on surface physicochemical properties and interactions with rabbit articular cartilage chondrocytes.Biomaterials.2005(2);26:3537-48.
33 Wang YW. Mo WK, et al. Biodegradation studies of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Polym Degrad Stability.2004(2);85:815-22.
34 Zhao K, Deng Y, et al. Polyhydroxyalkanoate (PHA) scaffolds with good mechanical properties and biocompatibility.Biomaterials.2003;24(13):1041-5.
35 Wang YW, Wu Q, et al. Reduced mouse cell growth by increased hydrophilicity of microbial polyhydroxyalkanoates via hyaluronan coating. Biomaterials.2005(7);24:4621-9.
36 Yang M, Zhu SS et al. Studies on bone marrow stromal cells affinity of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate). Biomaterials.2006;25(9):1365-73.