| ObjectiveAtherosclerotic 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.MethodsMale 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.Results1. HUVECs Isolation, culture and identification3d 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 identificationBMSCs 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 surfaceIn 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 flats4.MTT assay HUVECs, BMSCs proliferation condition on the material surfaceVertical 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.Conclusions1. 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.
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