应用组织工程材料栓塞动脉瘤的实验研究
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摘要
目的:探讨壳聚糖-甘油磷酸钠-成纤维细胞水凝胶作为颅内动脉瘤栓塞材料的可行性。为组织工程方法治疗动脉瘤提供实验依据。
方法:利用介入法结合弹力蛋白酶诱导兔颈总动脉,建立兔动脉瘤模型;用酶消化法从兔颈部皮肤中原代分离培养成纤维细胞,确定Brdu标记方法;配制2%(w/v)壳聚糖与56%(w/v)甘油磷酸钠溶液,不同比例混合,测定混合液pH、37℃成胶时间及力学强度,体外观察兔皮肤成纤维细胞在不同配比壳聚糖-甘油磷酸钠水凝胶中生长状态,选择既适合导管内操作又适合成纤维细胞生长的最佳比例;将Brdu标记的成纤维细胞与壳聚糖-甘油磷酸钠水凝胶混合栓塞兔颈动脉,评价组织相容性。最终栓塞兔动脉瘤模型。
结果:
(1)利用介入法结合弹力蛋白酶诱导兔颈总动脉,建立兔动脉瘤模型,4周时造影,观察动脉瘤宽径平均4.6±0.64mm;动脉瘤长径平均7.2±1.2mm;载瘤动脉即右侧头臂干直径平均4.0±0.72mm。
(2)胶原酶消化法及皮片接种法均可获得原代成纤维细胞,其中胶原酶消化法获得种子细胞效率高,更适合组织工程快速大量获得种子细胞的要求。胶原酶消化法在4小时时获得的有活性细胞量最多。Brdu可用于兔成纤维细胞标记,最佳标记方法为5ug/ml标记24小时。
(3)当壳聚糖/甘油磷酸钠在7:1时,pH值为7.28,成胶时间为260±18s,力学强度可达14KPa,细胞生存率最高,达(89±2.74)%。体外栓塞玻璃管动脉瘤模型见壳聚糖-甘油磷酸钠经过微导管原位成胶。
(4)动物体内实验:颈动脉栓塞,透视下可见混合物显影良好,局部组织切片显示(HE染色):1周时凝胶中散在较多细胞及钽粉,凝胶与血管壁边界较清,内皮细胞完整。4周时凝胶与血管壁边界不清,可见周边凝胶被细胞成分包绕降解。免疫荧光显示:4周标本中可见较多标记的成纤维细胞。兔动脉瘤栓塞术中见动脉瘤不显影,术后3d病理见动脉瘤被凝胶栓塞,内膜细胞形态正常,动脉壁内未见明显炎性细胞侵润。
结论:
本研究通过优化壳聚糖-甘油磷酸钠配比,构建壳聚糖-甘油磷酸钠-成纤维细胞水凝胶材料,利用这种材料进行兔颈动脉及动脉瘤模型栓塞。短期观察,证实壳聚糖-甘油磷酸钠-成纤维细胞水凝胶可用于动脉瘤栓塞。为组织工程方法治疗动脉瘤提供实验依据。
Intracranial aneurysms is the main reason of SAH, its incident rate is about 2% in natural crowd. With the high morbidity and mortality rates, this disease seriously threatens the healthy of people. There are two main ways to therapy aneurysms, they are clip operation and interventional therapy. With the development of catheter technique and embolism materials, interventional therapy has more and more been a main means. However ,some problems of interventional therapy gradually appear with the increasingly clinical application.. It has higher recanalization and recurrence rate of aneurysms , especially to giant or broad neck aneurysms. How to resolve this problem promotes the development of embolism technique and materials. Now a new technique-intravascular tissues engineering has been introduced to the strategy of intracranial aneurysms therapy. This way uses coils and stents as mechanical carrier, uses adenovirus、retrovirus or biodegradation high polymer materials as biology carrier, puts vitro prepared protein、gene、seed cells、extracellular matrix or cytokine into aneurysms cavity by common interventional technique, so as to promote the anatomy healing of aneurysms. There are some obstacles in the technique though, this assume has been approved by some experimental evidence.
Enlighten by the elementary way of the tissues engineering, we assume that combining tissue engineering technique and interventional technique, we can import three element of tissue construction (cell、biodegradation material、regulatory factor) into aneurysms through interventional means, make this material solidify hydrogel and block the aneurysms. Hydrogel provides three diamensions circumstance in which cells can co-attach、grow、propagate and secrete extracellular matrix, even to form tissue. As the same time extracellular frame degrades gradually and disappears. In the end ,tissue can occlude the aneurysms, its surface can be covered by vascular endothelial cell, so as to aneurysms permanent occlusion can be obtain by neoformative self tissue.
The objective of this study is to evaluate the feasibility of the injectable thermosetting chitosan–glycerophosphate-fibroblast (C-GP-FB) hydrogel as an embolism material of intracranial aneurysms. To try to construct a bioactive、injectable、thermosetting and bio-degraded hydrogel material, to embolism aneury- sms model, and to provide experimental evidences on how to use a new type liquid tissue engineering material to treat aneurysms.
In this study, firstly the rabbit aneurysms models were created ; Secondly , the fibroblasts were isolated and cultured from rabbit skin, and the way how to use Brdu to label rabbit fibroblasts was confirmed; Then C-GP hydrogel match was optimized and C-CP-FB material was constructed; Finally experiments and observation about using C-GP-FB embolism rabbit CA and aneurysms models were done. The tentative confirmation was that C-GP-FB material can be used to embolism the aneurysms.
1 The creation of rabbit aneurysms model
Methode:The rabbit as experimental animals. The first,we contrasted intra- arterial digital subtraction angiography and intra-venous digital subtraction angiography(DSA). Then we created rabbit aneurysms mode .The right common carotid artery was surgically exposed in all rabbits. Using endovascular techniques, we occluded the origin of the right common carotid artery with a Hyper Glide balloon. Elastase was incubated endoluminally in the proximal common carotid artery above the balloon in experiment group and PBS was in control group. The common carotid artery was ligated distally. Aneurysms’length、wildth and histology were obversed.
Results: (1)The best intra-venous DSA image was obtained when we choosed volume dose of contrast 4ml, inject speed 3ml/s, pressure 300, X-ray delay 4s,.(2)Saccular aneurysms formed in nine of the ten rabbits in experiment group. The width, height of aneurysms was 4.6±0.64mm and 7.2±1.2mm respectively. The rate of creation was 90%. Histology showed obliteration of the elastic lamina within the wall of the aneurysmal cavity. Aneurysms weren’t observed in the control group.
Conclusion: Stable saccular aneurysm model in the Rabbit can be created by using endovascular technique and elastase. Intra-venous DSA not only can show the aortic arch and upper-arcus artery clearly but also can be done repeatedly, and can be as an additional method to take place arteriographic.
2 Primary culture and labeling of rabbit skin fibroblast
Method:The fibroblasts were isolated and cultured from rabbit skin of neck using collagenase digestion and free skin graft inoculation. The cells were purified and identified, increasing curve of cells was drawn. Different labeling ratio of the fibroblast (3rd-5th passage) was observed under different label time and label dose in vitro.
Results:In collagenase digestion, the ratio of living cell decreased when collagenase time lengthened. The quantity of cells was explosion during 4-6 h, the most quantity of active cells was obtained in 4th h. Both labeling time and dose of BrdU determined labeling rate, but labeling time played more important role. The ratio reached to 94.5 with 5μg/mL BrdU incubating for 24 hours ,and this ratio wasn’t increased even if dose of Brdu was enlarged or labeling time was prolonged. Conclusion: 1、The fibroblasts can be isolated and cultured using collagenase digestion as well free skin graft inoculation., but the collagenase digestion means can obtain more cells and more fit for requirement of tissue engineering. 2、The most quantity of active cells was obtained in 4th h.. 3、Fibroblast can be purified by enzyme digestion and different adherence time. 4、The fibroblast from rabbit skin can stably passage 11 generation and has no morph variation. 5、Brdu can be used to label rabbit fibroblast. The optimal method is 5μg/mL Brdu incubating for 24 hours.
3 The optimization of C-GP hydrogel
Method:2% chitosan solution and 56% glycerophosphate solution were mixed in different volume ratio, these mixed solution were detected pH, colloidization time in 37℃,mechanic strength respectively. The cell activity was observed when the fibroblasts were culture in different ratio C-GP hydrogel. From above all, we determined the best ratio for which fit interventional operation as well as fibroblast grow.
Results: When the ratio of C/GP was 7:1 at pH 7.28, the colloidization time was 260±18 sec and the mechanic strength reached 14KPa and the cell survival rate reached peak 89±2.74% . In the experiment of embolizing aneurysms model in vitro, C/GP gel could arrive in aneurysms through catheter and made solid state.
Conclusion: 1、C-GP hydrogel has thermosetting character, under physiol- ogical pH and 37℃, the colloidization time can be control, this hydrogel has mechanical strength in some grade. 2、C-GP hydrogel has wonderful compatibility of cell and tissue. 3、When the ratio of C/GP was 7:1 at pH 7.28, the colloidization time in 37℃was about 4min and the mechanic strength reached 14KPa and the cell survival rate reached 89%. This C-GP hydrogel can be used to embolism aneurysms as frame material which carries cells or biomacromolecules.
4 The experimental observation of FB-C-GP hydrogel in vivo
Method: CCA of rabbit was isolated and ligated at distal. The fibroblasts labeling Brdu were mixed with C-GP in low temperature , then this C-GP-FB hydrogel was injected in CCA at proximal part, histocompatibility was evaluated. The rabbits which had been created aneurysms were exposed both femoral artery(FA), micro catheter and balloon were insert into FA respectively. Under the protection of balloon, C-GP-FB hydrogel was injected into aneurysms through micro catheter. The control of intra-catheter was evaluated , effect of embolism was observed.
Results: In the experiment of embolizing rabbit carotid arteries, C-GP-FB gel had fine visualization under X-ray. Histological section(HE stain) showed that: in 1st week , there were many cells in the gel, the boundary between gel and vessel wall was clear, endotheliocyte was eumorphism. In 4th week, the boundary between gel and vessel wall was obscure, slight degradation could be observed in the edge of hydrogel . Immumofluorescence showed that there were many labelled cell in the gel in 4th week. In the embolism operation of rabbit aneurysms, the aneurysms disappeared in angiography. In the HE stain slice 3 days after operation,C-GP-FB hydrogel in the aneurysms cave and endomembran eumorphism were observed,and there were no significant flame cell in the aneurysms wall.
Conclusion:The fibroblasts implanted C-GP-FB hydrogel can survive in rabbit CCA. Immigration of host cell in hydrogel can be observed. 2、C-GP-FB hydrogel can degrade in rabbits. 3、It is experimental testing that C-GP-FB hydrogel has feasibility through micro-catheter and stability in aneurysms. In the short term view, C-GP-FB hydrogel could be used to embolize aneurysms.
方法:利用介入法结合弹力蛋白酶诱导兔颈总动脉,建立兔动脉瘤模型;用酶消化法从兔颈部皮肤中原代分离培养成纤维细胞,确定Brdu标记方法;配制2%(w/v)壳聚糖与56%(w/v)甘油磷酸钠溶液,不同比例混合,测定混合液pH、37℃成胶时间及力学强度,体外观察兔皮肤成纤维细胞在不同配比壳聚糖-甘油磷酸钠水凝胶中生长状态,选择既适合导管内操作又适合成纤维细胞生长的最佳比例;将Brdu标记的成纤维细胞与壳聚糖-甘油磷酸钠水凝胶混合栓塞兔颈动脉,评价组织相容性。最终栓塞兔动脉瘤模型。
结果:
(1)利用介入法结合弹力蛋白酶诱导兔颈总动脉,建立兔动脉瘤模型,4周时造影,观察动脉瘤宽径平均4.6±0.64mm;动脉瘤长径平均7.2±1.2mm;载瘤动脉即右侧头臂干直径平均4.0±0.72mm。
(2)胶原酶消化法及皮片接种法均可获得原代成纤维细胞,其中胶原酶消化法获得种子细胞效率高,更适合组织工程快速大量获得种子细胞的要求。胶原酶消化法在4小时时获得的有活性细胞量最多。Brdu可用于兔成纤维细胞标记,最佳标记方法为5ug/ml标记24小时。
(3)当壳聚糖/甘油磷酸钠在7:1时,pH值为7.28,成胶时间为260±18s,力学强度可达14KPa,细胞生存率最高,达(89±2.74)%。体外栓塞玻璃管动脉瘤模型见壳聚糖-甘油磷酸钠经过微导管原位成胶。
(4)动物体内实验:颈动脉栓塞,透视下可见混合物显影良好,局部组织切片显示(HE染色):1周时凝胶中散在较多细胞及钽粉,凝胶与血管壁边界较清,内皮细胞完整。4周时凝胶与血管壁边界不清,可见周边凝胶被细胞成分包绕降解。免疫荧光显示:4周标本中可见较多标记的成纤维细胞。兔动脉瘤栓塞术中见动脉瘤不显影,术后3d病理见动脉瘤被凝胶栓塞,内膜细胞形态正常,动脉壁内未见明显炎性细胞侵润。
结论:
本研究通过优化壳聚糖-甘油磷酸钠配比,构建壳聚糖-甘油磷酸钠-成纤维细胞水凝胶材料,利用这种材料进行兔颈动脉及动脉瘤模型栓塞。短期观察,证实壳聚糖-甘油磷酸钠-成纤维细胞水凝胶可用于动脉瘤栓塞。为组织工程方法治疗动脉瘤提供实验依据。
Intracranial aneurysms is the main reason of SAH, its incident rate is about 2% in natural crowd. With the high morbidity and mortality rates, this disease seriously threatens the healthy of people. There are two main ways to therapy aneurysms, they are clip operation and interventional therapy. With the development of catheter technique and embolism materials, interventional therapy has more and more been a main means. However ,some problems of interventional therapy gradually appear with the increasingly clinical application.. It has higher recanalization and recurrence rate of aneurysms , especially to giant or broad neck aneurysms. How to resolve this problem promotes the development of embolism technique and materials. Now a new technique-intravascular tissues engineering has been introduced to the strategy of intracranial aneurysms therapy. This way uses coils and stents as mechanical carrier, uses adenovirus、retrovirus or biodegradation high polymer materials as biology carrier, puts vitro prepared protein、gene、seed cells、extracellular matrix or cytokine into aneurysms cavity by common interventional technique, so as to promote the anatomy healing of aneurysms. There are some obstacles in the technique though, this assume has been approved by some experimental evidence.
Enlighten by the elementary way of the tissues engineering, we assume that combining tissue engineering technique and interventional technique, we can import three element of tissue construction (cell、biodegradation material、regulatory factor) into aneurysms through interventional means, make this material solidify hydrogel and block the aneurysms. Hydrogel provides three diamensions circumstance in which cells can co-attach、grow、propagate and secrete extracellular matrix, even to form tissue. As the same time extracellular frame degrades gradually and disappears. In the end ,tissue can occlude the aneurysms, its surface can be covered by vascular endothelial cell, so as to aneurysms permanent occlusion can be obtain by neoformative self tissue.
The objective of this study is to evaluate the feasibility of the injectable thermosetting chitosan–glycerophosphate-fibroblast (C-GP-FB) hydrogel as an embolism material of intracranial aneurysms. To try to construct a bioactive、injectable、thermosetting and bio-degraded hydrogel material, to embolism aneury- sms model, and to provide experimental evidences on how to use a new type liquid tissue engineering material to treat aneurysms.
In this study, firstly the rabbit aneurysms models were created ; Secondly , the fibroblasts were isolated and cultured from rabbit skin, and the way how to use Brdu to label rabbit fibroblasts was confirmed; Then C-GP hydrogel match was optimized and C-CP-FB material was constructed; Finally experiments and observation about using C-GP-FB embolism rabbit CA and aneurysms models were done. The tentative confirmation was that C-GP-FB material can be used to embolism the aneurysms.
1 The creation of rabbit aneurysms model
Methode:The rabbit as experimental animals. The first,we contrasted intra- arterial digital subtraction angiography and intra-venous digital subtraction angiography(DSA). Then we created rabbit aneurysms mode .The right common carotid artery was surgically exposed in all rabbits. Using endovascular techniques, we occluded the origin of the right common carotid artery with a Hyper Glide balloon. Elastase was incubated endoluminally in the proximal common carotid artery above the balloon in experiment group and PBS was in control group. The common carotid artery was ligated distally. Aneurysms’length、wildth and histology were obversed.
Results: (1)The best intra-venous DSA image was obtained when we choosed volume dose of contrast 4ml, inject speed 3ml/s, pressure 300, X-ray delay 4s,.(2)Saccular aneurysms formed in nine of the ten rabbits in experiment group. The width, height of aneurysms was 4.6±0.64mm and 7.2±1.2mm respectively. The rate of creation was 90%. Histology showed obliteration of the elastic lamina within the wall of the aneurysmal cavity. Aneurysms weren’t observed in the control group.
Conclusion: Stable saccular aneurysm model in the Rabbit can be created by using endovascular technique and elastase. Intra-venous DSA not only can show the aortic arch and upper-arcus artery clearly but also can be done repeatedly, and can be as an additional method to take place arteriographic.
2 Primary culture and labeling of rabbit skin fibroblast
Method:The fibroblasts were isolated and cultured from rabbit skin of neck using collagenase digestion and free skin graft inoculation. The cells were purified and identified, increasing curve of cells was drawn. Different labeling ratio of the fibroblast (3rd-5th passage) was observed under different label time and label dose in vitro.
Results:In collagenase digestion, the ratio of living cell decreased when collagenase time lengthened. The quantity of cells was explosion during 4-6 h, the most quantity of active cells was obtained in 4th h. Both labeling time and dose of BrdU determined labeling rate, but labeling time played more important role. The ratio reached to 94.5 with 5μg/mL BrdU incubating for 24 hours ,and this ratio wasn’t increased even if dose of Brdu was enlarged or labeling time was prolonged. Conclusion: 1、The fibroblasts can be isolated and cultured using collagenase digestion as well free skin graft inoculation., but the collagenase digestion means can obtain more cells and more fit for requirement of tissue engineering. 2、The most quantity of active cells was obtained in 4th h.. 3、Fibroblast can be purified by enzyme digestion and different adherence time. 4、The fibroblast from rabbit skin can stably passage 11 generation and has no morph variation. 5、Brdu can be used to label rabbit fibroblast. The optimal method is 5μg/mL Brdu incubating for 24 hours.
3 The optimization of C-GP hydrogel
Method:2% chitosan solution and 56% glycerophosphate solution were mixed in different volume ratio, these mixed solution were detected pH, colloidization time in 37℃,mechanic strength respectively. The cell activity was observed when the fibroblasts were culture in different ratio C-GP hydrogel. From above all, we determined the best ratio for which fit interventional operation as well as fibroblast grow.
Results: When the ratio of C/GP was 7:1 at pH 7.28, the colloidization time was 260±18 sec and the mechanic strength reached 14KPa and the cell survival rate reached peak 89±2.74% . In the experiment of embolizing aneurysms model in vitro, C/GP gel could arrive in aneurysms through catheter and made solid state.
Conclusion: 1、C-GP hydrogel has thermosetting character, under physiol- ogical pH and 37℃, the colloidization time can be control, this hydrogel has mechanical strength in some grade. 2、C-GP hydrogel has wonderful compatibility of cell and tissue. 3、When the ratio of C/GP was 7:1 at pH 7.28, the colloidization time in 37℃was about 4min and the mechanic strength reached 14KPa and the cell survival rate reached 89%. This C-GP hydrogel can be used to embolism aneurysms as frame material which carries cells or biomacromolecules.
4 The experimental observation of FB-C-GP hydrogel in vivo
Method: CCA of rabbit was isolated and ligated at distal. The fibroblasts labeling Brdu were mixed with C-GP in low temperature , then this C-GP-FB hydrogel was injected in CCA at proximal part, histocompatibility was evaluated. The rabbits which had been created aneurysms were exposed both femoral artery(FA), micro catheter and balloon were insert into FA respectively. Under the protection of balloon, C-GP-FB hydrogel was injected into aneurysms through micro catheter. The control of intra-catheter was evaluated , effect of embolism was observed.
Results: In the experiment of embolizing rabbit carotid arteries, C-GP-FB gel had fine visualization under X-ray. Histological section(HE stain) showed that: in 1st week , there were many cells in the gel, the boundary between gel and vessel wall was clear, endotheliocyte was eumorphism. In 4th week, the boundary between gel and vessel wall was obscure, slight degradation could be observed in the edge of hydrogel . Immumofluorescence showed that there were many labelled cell in the gel in 4th week. In the embolism operation of rabbit aneurysms, the aneurysms disappeared in angiography. In the HE stain slice 3 days after operation,C-GP-FB hydrogel in the aneurysms cave and endomembran eumorphism were observed,and there were no significant flame cell in the aneurysms wall.
Conclusion:The fibroblasts implanted C-GP-FB hydrogel can survive in rabbit CCA. Immigration of host cell in hydrogel can be observed. 2、C-GP-FB hydrogel can degrade in rabbits. 3、It is experimental testing that C-GP-FB hydrogel has feasibility through micro-catheter and stability in aneurysms. In the short term view, C-GP-FB hydrogel could be used to embolize aneurysms.
引文
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