| Background:Intracranial aneurysm (ICA) refers to the limitations of cystic dilatation causing by weak intracranial arterial wall, and is the most common reason of spontaneous subarachnoid hemorrhage. Traditionally, as a serious threat to human health, aneurysms were found mostly due to spontaneous subarachnoid hemorrhage caused by aneurysm rupture, and its course is hide and sudden-onset. Once the aneurysm rupture and bleed, the mortality rate was as high as 25% to 40% and the rate of disability was about 33%. Moreover, re-bleeding mortality can reach up to 40% to：实验结果显示负载SDF-1α弹簧圈栓塞+EPCs移植组的动脉瘤颈为新ofintracranial aneurysm clearly should be paid sufficient attention and we must take active treatment for the patients with high risk of rupture to prevent aneurysm rupture. Currently, the main treatments for aneurysm include microsurgery surgical clipping and endovascular embolization, and the endovascular embolization has incomparable superiority for the brain damage caused by surgical operation. Moreover, to aneurysm ruptured, endovascular embolization can significantly reduce mortality and patient compliance is well. And after the treatment of endovascular embolization, the treatment of endovascular embolization, the absolute risk fall 7.4% and result in a lower probability of seizures. But both the spring coil current commonly used in clinical lacking of biological activity and the lack of complete endothelialization at the aneurysm neck after embolism aneurysm can lead to residual or recurrence of the aneurysm. To solve this problem, in addition to searching for new material or designing different kinds of complicated shape spring coil, biological functionalization of platinum micro spring coil can play a multiplier effect.Since the Italian scholar Guglielmi (1989) designed Guglielmi detachable coils (GDC), the endovascular embolization techniques using internal platinum coil with advantage of minimally invasive, quick recovery and fewer complications become a preferred option in some of the international center for the treatment of intracranial aneurysms. The main mechanism of spring coil applied in aneurysm endovascular embolization treatment is mainly through platinum coil mechanical embolization Endovascular aneurysm mainly through mechanical tamponade. And then block tumor cavity and change the local hemodynamic factors to eliminate the risk of aneurysm rupture and bleeding. However, the standard platinum GDC existing biologically inert, aneurysms after embolization is difficult in thrombose, thrombus orgniztion and can’t resist the persistent arterial pulse and recanalization. Therefore, compared with the conventional surgical craniotomy clipping, endovascular aneurysm is still having higher recurrence rate. In some large-scale international clinical research, follow-up through digital subtraction angiography (DSA) found that the recurrence rate of treatment using platinum coil is more than 20% and some patients is still bleeding again in the future. To compensate for the current shortage of GDC and accelerate the aneurysm cavity fibrosis and the endothelial coverage at aneurysm neck, such as the platinum coil coating biological absorbable polymer loaded with some cell activation factor used in treatment. Thus, with the degradation of the polymer material and controlled release, the biological activity may be improved and more conducive to make up the aneurysm embolization postoperative healing of anatomy and reduce the risk of recurrence and rebleeding.In recent years, studies have shown that:chemokines in protecting the host, clearing pathogenic factor, inflammatory response and immune system homeostasis, etc, and the development and repair of damaged areas of cardiovascular and cerebrovascular systems have played an important role. Chemokines is a class of chemotaxis that can regulate cytokine cell migration, and their receptors are GTP protein coupling transmembrane receptors that mediated chemokines exercising its biological function. Usually, they expressed on the membrane of immune cells and endothelial cells. Stromal cell-derived factor-la (SDF-la) is a kind of chemotactic factor as a member of the CXC subfamily and can combine with its receptor CXCR4 in the cell surface to come into play its role specific. Numerous studies have indicated that SDF-la and its receptor were involved in regulating the migration of a variety of nerve cells during the development of central nervous system. Recent studies have shown that the more primitive EPCs can express SDF-la receptor CXCR4, thus SDF-la can chemotaxis EPCs and promoting their migration, homing and differentiate into endothelial cells locally.Endothelial progenitor cells (EPCs) is a kind of precursor cells, which is similar to hemangioblastoma that can proliferate, differentiate into mature endothelial cells. Under physiological conditions, it is mainly engaged in the maintenance and reconstruction of vascular dynamics. However, under pathological conditions, when the compensatory of organism is difficult to generate sufficient repair capacity, especially in the patients with risk factors of ischemic cardiovascular disease, the number and migration ability of EPCs were significantly decreased. Studies have shown that the correlation of the number of EPCs and Framingham cardiovascular risk index was negative. This suggests that enriched, amplified EPCs in vitro and autologous transplant is a positive and effective strategy to facilitate vascular repair or regeneration. EPCs can also act as a good source of seed cells in tissue engineering reconstruction of vascular structure. The mobilization and transplantation of EPCs can improve the ability of angiogenesis in ischemic tissue and promote blood vessel repair and re-endothelialization. There also have amount of reports that EPCs transplantation was used to treat a variety of vascular damage models, such as cerebral ischemia, limb ischemia, myocardial infarction, carotid endarterectomy, etc. This study is aimed to construct a novel bioactive coils coated with SDF-la that can release SDF-la sustained. And when the coil pack artery aneurysm rat model, it can promote migration and homing of EPCs and the proliferation of aneurysm endothelial, aneurysm cavity fibrosis and endothelial coverage at aneurysm. All of the above function will reduce the recurrence rate after embolization. It is one of the content of this research that how the SDF-1a can be coated on surface of the platinum micro coil and released controlled and sustained.In the present study, silk fibroin (SF), a natural protein that is a biomaterial including many advantages such as excellent biocompatibility, biodegradable was used in tissue engineering research. It has the properties including induced to β-sheet formation, changeable solubility in aqueous solution from soluble to insoluble. According to this feature, we use silk fibroin as the transmission system of SDF-la and coated it on the surface of coils. The SDF-la will be released sustained by the change of silk fibroin solubility property.In summary, the effective load of SDF-la, sustained release and controlled absorption will be achieved by the use of biocompatible, absorbable silk fibroin-coated platinum coil. By the way, the SDF-la binding to its receptor CXCR4 can promote the migration and homing of EPCs, the proliferation of aneurysm endothelial, fibrosis of, and aneurysm cavity and the embolization at the aneurysm neck. Thus, the aneurysm embolization postoperative healing of anatomy can be made up and the risk of recurrence and rehaemorrhagia.Part I Preparation of bioactive coils coated with SDF-la and the structural characterization and property of the modified microcoilObjective:This part study is to preparing a new type of biological active coils coated SDF-la and detect the structural characterization and the property of releasing SDF-la sustained. It is the foundation of SDF-la-coated coils together with EPCs recruitment to promote the healing of aneurysm neck.Materials and Methods:Preparing coils in length of about 8 mm and draining by pumps after ultrapure water. 10μg of SDF-la was dissolved in 500μl of 3%(m/v) silk fibroin solution and dried coil was immersed in the above solution to construct a SDF-la-coated coils. Coil morphologies before and after coating with SDF-la were observed under a scanning electron microscope (SEM). The sustained releasing of SDF-la-coated coils was detected by enzyme-linked immunosorbent assay (ELISA). Results:The results by SEM showed that the surface of treated coils is uniformly and the change is little compared with untreated. In the test cycle of released quantity, SDF-la-coated coils were showed a significant accumulation increasing trend. Conclusions:SDF-la-coated coils were successfully produced. The coils were loaded SF/SDF-1a in uniform and released SDF-1a under control in vitro.Part II Separation, culture, identification of EPCs and the promoting migration by SDF-la-coated coilsObjective:EPCs were separated, cultured and identificated from rat bone marrow. And through the experiment in vitro, coated coils can be demonstrated playing an acceerative role in EPCs migration. All the above provide a foundation to study the influence on the healing of aneurysm neck by coated coils in EPCs recruitment.Materials and Methods:Sprague-Dawley rats (weighing about 100g) bone marrow was obtained from the long bones. Mononuclear cells were isolated using Ficoll-PaqueTM lymphocyte separation and induced to differentiate EPCs by endothelial growth medium-2MV (EGM-2MV). And then observe theirs morphological feathers. Flow Cytometer (FCM) was used to detect the percentage of positive expression. Capillary-like tube formation assay was applied to test the ability of EPCs. Transwell migration assay was observed the influence of SDF-la-coated coils on EPCs migration.Results:Mononuclear cells began to adhere to after cultured 24-48h and were in typical spindle morphology after cultured 3-7d. "Cord-like structures" and "Network formation" were emerging during 7-14d. The cells changed into typical cobblestone on day 28. Flow cytometry analysis showed that the percentage of cell surface markers CD133, CD34 and vascular endothelial growth factor receptor-2 (VEGFR-2) were as follows:66.38%,88.87%,82.69%. Capillary-like tube formation assay indicated that EPCs possess the ability of was applied to test the ability of EPCs. Transwell migration assay demonstrated that the migratory function of EPCs would be elevated by SDF-la-coated coils.Conclusions:Bone marrow mononuclear cells were isolated from bones of rats by density gradient centrifugation and cultured in endothelial growth medium-2MV (EGM-2MV) supplemented with vascular endothelial growth factor (VEGFR-2), basic human fibroblast growth factor (bFGF). We obtained more purified early EPCs by flow cytometry to analyze phenotypic characterization of EPCs after staining with anti-CD34, anti-VEGFR-2 (KDR), and anti-CD 133.Tube formation assay played the same result. Transwell migration assay indicated that SDF-la-coated coils elevate the migratory function of EPCs.Part Ⅲ SDF-la-coated coils accelerates intra-aneurysmal organization and occlusion of neck remnant by recruiting EPCsObjective:To track the relation between transplanted cells and SDF-la-coated coils and clarify the function of SDF-la-coated coils to wound healing and repair to rats aneurysm neck, the rat aneurysm model was built.Materials and Methods:A total of 60 male rats were randomly divided into five groups:sham-operated group, unmodified coils group, unmodified coils+ transplantation of EPCs group, SDF-la-coated coils group, and SDF-la-coated coils +transplantation of EPCs group. Each of the coils was inserted into the right common carotid artery (CCA) according to the different sets. EPCs suspension was infused into tail vein immediately after coil implantation. On day 14 and 28 after coil implantation, rats from each group were sacrificed. CCA segments containing the coils were collected and fixed. And the aneurysm neck morphologies were observed under a standard light microscope. HE staining, Van Geson staining and Verhoeff’s staining, immunofluorescence staining were used for doing pathology examination to aneurysm tissue. Cultured EPCs attaching on coils were detected by using fluorescence microscopy. SEM was used to detect attached cells on surface of coils.Results:The results showed that aneurysm healing of SDF-la-coated coils and transplantation of EPCs group was obvious better than other groups (P<0.05) on day 14,28. On day 14, the number of cells attached on SDF-1α-coated coils and transplantation of EPCs group was more than all the other groups. SDF-la-coated coils and transplantation of EPCs group and SDF-la-coated coils group can be observed the coverage of fibrosis on day 28, but there were only some cell cluster in other three groups. Pathological examination of aneurysm tissue by HE staining, Van Geson staining and Verhoeff’s staining, immunofluorescence staining reveal that SDF-la-coated coils and transplanted EPCs can accelerate endothelial cell proliferation and aneurysm fibrosis to the goal of cavity occlusion.Conclusions:Combined treatment with SDF-la-coated coils and EPCs transplantation could more effectively chemotaxis EPCs, accelerate endothelial cell proliferation and aneurysm fibrosis, repairing and reconstruction of the aneurysm neck. This may provide a new strategy for promoting aneurysm healing and preventing the recurrence of aneurysm. |
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