Introduction: Stenting attracts attention as a low-invasive operation for aneurysm or stenosis. However restenosis is generated after stenting as a main problem. Researchers show the vascular intima is injured and smooth muscle cells (SMC) multiply excessively after placement of stent. And then restenosis may be happened.
Kipshidze et al[1]. indicate that endothelial cells (EC) suppress the exuberant of SMC. In the previous study by Grewe et al[2]., EC start to multiply after SMC growing and become neointima. Then, we can say restenosis will be caused by slow of endothelialization. This assumption can lead quick endothelialization is important for the suppression of restenosis.
To lead quick endothelialization, the type I collagen, one of the extracellular matrix, immobilized on wire may promote the endothelialization because collagen has an effect to promote of cellular adhesion[3].
Kobayashi et al[4]. developed a method which can immobilize collagen on the material surface using HMDI. This method immobilizes collagen firmly by covalent bond and coats collagen uniformly.
Material and Method: Immobilization of Type Ⅰ collagen on the wire: 0.406 mm × 0.406 mm cross-section NiTi wire was cleaned for 20 minutes by the UV-O3 cleaning system. Then, the wire was put in the toluene solution including 10 vol% HMDI and 0.01 vol% dibutyltin diraulate for 30 minutes in nitrogen gas atmosphere. After that, the wire was rinsed with acetone and soaked in 5 mg/ml Type Ⅰ collagen solution for an hour. Unreacted solution was removed by rinsing with 0.01M HCl, and DDW.
Incubation: Human Carotid Artery EC (P5~8) was cultured on gelatin coated dishes for 3 days to be a confluent state in an incubator (37℃, 5%CO2). After that, we placed a bare wire or the collagen immobilized wire on the dishes and incubated the models for 24 hours.
After the incubation, the wire was taken out from the model, and fixed with 4% paraformaldehyde, then incubated with Alexa Fluor 546 Phalloidin and DAPI.
Result and Discussion: Representative fluorescent images on wire are shown in Fig. 1.
The expression of F-actin that plays important role in cell adhesion, shows the firm adherence. And the cell density on wire corresponding to the images are also shown Fig. 2 as a quantitative results.
The cell density on the side surface with collagen is much higher than that of bare.
If the side surface of wire is divided into 4 sections from the bottom to the top, the lower the section has the higher the cell density. The results show that the endothelialization on the wire was performed by cells climbing up from the culture surface.
Concluding Remarks: To evaluate the endothelialization on wire, EC density on stent was observed and measured. The wire with Collagen has more EC than the bare wire even if the flow chamber. The result suggests that the immobilization of collagen could promote the quick endothelialization.
JSPS Grant-in-aid Scientific Research (B), (25282140)
References:
[1] N.Kipshidze，G.Dangas，M.Tsapenko，J.Moses，M.B.Leon，M.Kutryk，P.Serruys， “Role of the Endothelium in Modulating Neointimal Formation. Vasculoprotective Approaches to Attenuate Restenosis After Percutaneous Coronary Interventions” ，Journal of the American College of Cardiology，2004; 44: 733-739
[2] P.H.Grewe，T.Deneke，S.K.Holt，A.Machraoui，J.Barmeyer，K.M.Muller， “Scanning electron microscopic analysis of vessel wall reactions after coronary stenting”，Z Kardiol，2000; 89: 21-27
[3] M.L.Lu，R.J.Mccarron，B.S.Jacobson，“Initiation of HeLa cell adhesion to collagen is dependent upon collagen receptor upregulation, segregation to the basal plasma mambrane clustering and binding to the cytoskeleton”，Journal of Cell Science，1992; 101: 873-883
[4] H.Kobayashi，Y.Ikeda，“Covalent immobilization of proteins on to the surface of poly (vinyl alchol) hydrogel”，Biomaterials，1991; 12: 747-751