构建去细胞牛颈静脉内皮化实验研究
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摘要
目的：研究去细胞加光氧化交联处理后牛颈静脉血管片表面改性方法,增加组织工程血管材料内皮细胞的黏附生长。
方法：选取新鲜合适的牛颈静脉,采用多步骤去垢剂-酶消化法脱细胞和光氧化交联,经处理后的牛颈静脉脱细胞基质是比较理想的组织工程血管支架。应用纤维连接蛋白(Fibronectin, Fn),Ⅳ型胶原(Collagen, ColⅣ),明胶为单独预衬成分,纤维连接蛋白+明胶,纤维连接蛋白+Ⅳ型胶原+明胶为混合预衬成分,设置磷酸缓冲液体组(PBS)预衬为对照组,每组成分均以30μl的剂量采用物理包裹法,预衬去细胞牛颈静脉血管片54片(裁剪每片面积约1.6cm2)。每片种植人脐静脉内皮细胞株细胞(CRL-2480)5×105,静态培养7天,取1,4,7天标本消化血管片表面细胞记数,标本切片HE染色,扫描电镜和透射电镜检测,比较各处理组血管片表面细胞黏附数目,结合程度,对比不同预处理方法对增强细胞黏附性和促进生长情况的影响,探索最佳组织工程血管改性技术。
结果：(1)细胞记数：细胞培养第1天,预衬组细胞数目多于对照组(p<0.05),预衬处理组间无明显差别。第4-7天,混合预衬组与单一预衬组间有差别(p<0.05),混合预衬组内无差别(p>0.05)。(2)标本石蜡包埋后切片HE染色结果：第1天各组血管片表面细胞排列密集,紊乱,细胞核有堆积,部分呈双层排布；第4天,对照组血管表面细胞疏松,有成片分布；预衬组细胞连接成片,呈单层排布。第7天,对照组和单独预衬组细胞都较少,混合预衬组细胞铺连成片,与下方纤维结构有连接,未出现表皮脱落。(3)取7天标本扫描电镜显示：PBS组血管片表面细胞稀疏,细胞间无桥接,与下方的纤维结构联结不明显。混合预衬组铺连成片,细胞间长梭形有桥接和突起,与去细胞的血管片纤维之间形成连接。(4)透射电镜：可观察到表面生长内皮细胞与下方纤维成分有空白带存在。
结论：去细胞加光氧化处理后的牛颈静脉血管片可生长内皮细胞。经过Fn, ColⅣ,明胶单独预衬处理的血管片表面,可增强内皮细胞黏附和贴壁生长。混合预衬比单独预衬处理在增强细胞黏附性及贴壁生长方面效果更好。
目的：从犬骨髓细胞中分离出骨髓间充质干细胞,提取内皮祖细胞,体外培养分化为内皮细胞,种植到混合预衬去细胞牛颈静脉材料上培养,体外构建组织工程血管。
方法：1.使用Ficoll淋巴细胞分离液,通过密度梯度离心法从犬骨髓细胞中分离出单个核细胞,骨髓单个核细胞通过反复贴壁培养法获得内皮祖细胞,CD34抗原染色鉴定；含多种细胞生长因子的内皮细胞培养基培养,促进内皮祖细胞分化为成熟的内皮细胞,CD31和人类Ⅷ因子相关抗原染色鉴定。
2.混合预衬材料处理牛颈静脉脱细胞血管材料,将内皮细胞种植到血管材料,体外静态培养7天后,HE染色观察表明细胞覆盖情况及扫描电镜检测血管片上细胞的生长情况。
结果：原代培养的骨髓间充质干细胞呈螺旋状、放射状生长排列。原代培养的骨髓内皮祖细胞很快形成克隆,第2代细胞中CD34相关抗原的表达阳性率约45%,CD31和Ⅷ因子表现阴性。从第4代细胞起表现出内皮细胞样形态,呈“铺路石”状,并表现接触抑制的特点,第5代细胞CD34相关抗原表达阴性,CD31和Ⅷ因子相关抗原表达的阳性率为78%左右。种植于组织血管表面细胞,HE染色显示细胞覆盖血管表面,扫描电镜检测到血管片上均有较致密的细胞覆盖生长,但细胞排列无序。
结论：骨髓来源的间充质干细胞分化为内皮细胞,可种植于脱细胞牛颈静脉血管,构建内皮化组织工程血管。
目的：将种植内皮细胞的组织工程血管,用于重建犬右心室流出道实验模型,观察体内组织工程血管与血液接触的条件下的表面内皮化、细胞浸润以及钙化、血栓情况,评价内皮化去细胞牛颈静脉临床应用可行性。
方法：以种植分化扩增所获内皮细胞的去细胞牛颈静脉带瓣管道为研究对象,建立犬重建右心室—肺动脉连接的动物模型,设立未种植细胞的去细胞结合光氧化制备的牛颈静脉血管为对照组。实验动物分别于1月、3月和6月屠宰后取出标本,大体观察了解血管形态改变；组织化学和免疫组织化学染色观察各种炎性细胞浸润、表面内皮细胞生长,电镜检查了解植入后的管壁超微结构变化。
结果：17只犬除了5只死于手术中室颤外,余12只得以存活,其中对照组和试验组各6头。于1月、3月和6月各宰杀4只,对照组和试验组各2头。大体解剖标本：1月、3月对照组和试验组均无血栓和钙化形成,；6月对照组犬存在血栓沉着和近端吻合口膜样增生,存在血管部分钙化点,试验组均无异常。Ⅷ因子染色显示：1、3、6月时,试验组标本都表现出染色阳性,对照组只有6月的8因子染色结果显示为阳性。von kossa钙盐染色对照组在6月见到明显钙化点,试验组未见钙化点。电镜结果显示：试验组在1月时血管表面细胞部分覆盖,对照组无细胞。3月试验组标本表面细胞铺连成簇,对照组部分覆盖,可见裸露血管。6月试验组可见铺连成片细胞,对照组仍有部分组织工程血管显露。
结论：犬骨髓间充质干细胞分化为内皮细胞后,种植预衬处理后去细胞牛颈静脉血管,在重建右室流出道体内实验中表现出良好生长活性、抗钙化能力和较好内皮化程度。
Objective:To explore Surface Pre-coating method of decellularized Bovine jugular vessel sheets,to increase endothelial cells' biocompatibility,attachment and growth on the tissue Engineering material.
Methods:Pick out fresh and suitable bovine jugular vein as the tissue Engineering material.Acellular bovine jugular vein matrix is made through a gentle multi-process decellularization protocols and cross-linked by dye-mediated photooxidation.Apply Fibronectin, CollagenⅣ,gelatin as 3 single Pre-coating groups, Fibronectin and gelatin, Fibronectin and Collagen IV and gelatin as 2 mixtured Pre-coating groups, PBS liquid as the compared group.Six different compositions were deposed onto surface of 54 BJV sheets with a dose of 30μl,and each sheet,which was cut out at 1.6 cm2,was implanted with human umbilical vein endothelial cells(CRL-2480) 5×105 on surface. Then culture the sheet under static condition for 7 days.Took the sample at 1st,4th,7th day,depart cells from sheet surface with enzyme and count numbers.Took samples for HE stain and took photos on microscope.The 7th day samples of the compared and the mixtured-Pre-coating groups had scanning and transmission electron microscope checks.By comparing each groups'effect on increasing cell's biocompatibility,growth and disposition, explore the best surface pre-coating method of decellularized BJV sheets,so as to get the best endothelialization of BJV sheet.
Results:(1)Cell number:In 1st day of culture course,Pre-coating groups'surface cells,which is departed with enzyme,were more than compared group's(p<0.005).There was no significant difference between pre-coating groups.In 4th to 7th day,there was a significant difference between the single and mixture Pre-coating groups(p<0.05),the mixture Pre-coating groups had more cells.There was no obvious difference between the 2 mixture pre-coating groups.(2)All samples had a section after paraffin imbedding, then had a HE stain and took photos on microscope.At 1st day,each groups'surface had a cell layer,spread closely,cellular nucleus could see an accumulation.At 4th day,the compared group's surface cells had a decoherence,while the pre-coating group gained a cell layer well.At 7th day, the compared and single pre-coating group were scarce of cells, fibre below was partly uncoated.The mixtured pre-coating group's surface cells had formed a integrity layer,linked with fibre below. (3)Took the 7th sample for a scanning electron microscope.On the compared group's surface, the cells were scarce and no conjunction linked between cells or cell and sheet. The mixtured Pre-coating group had more cells.Cellular skeleton was formed and linked with vessel sheet fibre. Among cells, there was fusiform-shaped axon as linkage,which was a tight connection of cells and fibre. (4)Transmission electron microscope could see vacant brand among surface cells and fibre below BJC sheet.
Conclusion:Decellularized BJV sheets are capable of growing endothelial cells.Under Fn, ColⅣ,gelatin single modification, the sheet gain an augment of cells' biocompatibility, attachment and growth. Mixtured Pre-coating is better than single pre-coating on increasing cells' attachment and growth.
Objective:Bone marrow mesenchymal stem cells (MSCs) and endothelial progenitor cells(EPC) were separated from dogs'bone marrow and then incubated for differentiation into endothelial cells. The endothelial cells were implanted on decellularized bovine jugular vessel in order to form tissue-engineering vessels.
Methods:1.Dogs'bone marrow mononuclear cells(BM-MNCs) were separated by gradient centrifugation on Ficoll (density 1.077g/ml) from bone marrow and EPCs were collected through adhearance cultivateding repeatedly.Cells were identified by CD34 dyeing,then incubated with DMEM and factorsfor to help EPCs differentiate to normal endothelial cells which were identified by CD31 and VIII factor dying.
2.The normal endothelial cells were planted on bovine jugular vein matrixs in static condition for 7 days.Cells'coverage and cultivation were tested with HE dying.The morphologic structure was observed with photographed by scanning electron microscope (SEM).
Results:The adherent BM-MNCs grew in radiative and spiral way and were proved to be EPCs 45% of which showed positive for CD34 and negative for CD31 and factorⅧ-related antigen..Primary culture EPCs formed clones quickly.The 4th generation cells'morphologic structure were like endothelial cells',and contact inhibition was observed.78% of the 5th generation cells were positive for CD31 and factorⅢ-related and negative for CD34 antigen.The cells planted on the conduits'surface proved to be well cultivated over the surface by HE dying.And the SEM showed that the coverage on the surface was compact without proper order.
MSCs were incubated for orientated differentiation into smooth muscle-like cells which showed positive for a-SMA.
Conclusions:Bone marrow MSCs and EPc can be differented into ECs in vitro respectively. ECs can be successfully implanted on the acellular scaffolds of bovine jugular vein to built tissue-engineering vessels.
Objective:The conduits with cell plantation were used to reconstruct dogs' right ventricle outflow tract.This study was to investigate the histological change of endothelialization BJVCs and evaluate the possibility of which as a tissue engineering scaffold in clinical application.
Methods:Decelluarizated valved BJVCs with cell plantaion were used to rebuilt dogs'right ventricle outflow tract.The control group were decelluarizated valved BJVCs without cell platntaion.The conduits were explanted and analyzed by HE examination, light microscopy, and electron microscopy in 1 month,3 months and 6 months after being implanted. Tissue contents of collagen, elastin and glycosaminoglycans were assayed respectively to the explanted tissues.
Results:17 dogs subjected implantation,5 died of ventricular fibrillation in operation and the other 12 ones survived.4 dogs sacrifaced in 1 month after operation, another 4 dogs sacrifaced in 6 month and the others sacrifaced in 1 year. In 1st and 3nd month,the test and control group had no thrombus or calcification in Macroscopic observation.In 6th month, thrombus and ndomembrance proliferation was observed near the anastomotic stoma in the control group,and part of the conduits were calcificated.The test group was normal.FactorⅧ-related dying were observed positive in all time point in test group,while positive in 6th month in control group. Von kossa calcium dying were positive in 6th month vessel of control group,while nagtive in test group conduit of all time point.The Scanning electron microscope showed that cells were covered part of the test groups'surface,and the controul group scarce in 1st month.In 3th month,the test group gained more coverage of cells,and the control group gained partly.In 6th month,cells on the test group vessels were contacted tightly and like a plaque,but the control group were partly covered and fibers below was obvious.
Conclusions:Pre-coating decelluarizated valved BJVCs with cell plantaion have growth potentiality and supporting endothelialization, with low-immunogenicity and low-calcification, which surpport the conduit as a tissue engineering scaffold for reconstructing the connection of pulmonary arteries with right ventricles.
方法：选取新鲜合适的牛颈静脉,采用多步骤去垢剂-酶消化法脱细胞和光氧化交联,经处理后的牛颈静脉脱细胞基质是比较理想的组织工程血管支架。应用纤维连接蛋白(Fibronectin, Fn),Ⅳ型胶原(Collagen, ColⅣ),明胶为单独预衬成分,纤维连接蛋白+明胶,纤维连接蛋白+Ⅳ型胶原+明胶为混合预衬成分,设置磷酸缓冲液体组(PBS)预衬为对照组,每组成分均以30μl的剂量采用物理包裹法,预衬去细胞牛颈静脉血管片54片(裁剪每片面积约1.6cm2)。每片种植人脐静脉内皮细胞株细胞(CRL-2480)5×105,静态培养7天,取1,4,7天标本消化血管片表面细胞记数,标本切片HE染色,扫描电镜和透射电镜检测,比较各处理组血管片表面细胞黏附数目,结合程度,对比不同预处理方法对增强细胞黏附性和促进生长情况的影响,探索最佳组织工程血管改性技术。
结果：(1)细胞记数：细胞培养第1天,预衬组细胞数目多于对照组(p<0.05),预衬处理组间无明显差别。第4-7天,混合预衬组与单一预衬组间有差别(p<0.05),混合预衬组内无差别(p>0.05)。(2)标本石蜡包埋后切片HE染色结果：第1天各组血管片表面细胞排列密集,紊乱,细胞核有堆积,部分呈双层排布；第4天,对照组血管表面细胞疏松,有成片分布；预衬组细胞连接成片,呈单层排布。第7天,对照组和单独预衬组细胞都较少,混合预衬组细胞铺连成片,与下方纤维结构有连接,未出现表皮脱落。(3)取7天标本扫描电镜显示：PBS组血管片表面细胞稀疏,细胞间无桥接,与下方的纤维结构联结不明显。混合预衬组铺连成片,细胞间长梭形有桥接和突起,与去细胞的血管片纤维之间形成连接。(4)透射电镜：可观察到表面生长内皮细胞与下方纤维成分有空白带存在。
结论：去细胞加光氧化处理后的牛颈静脉血管片可生长内皮细胞。经过Fn, ColⅣ,明胶单独预衬处理的血管片表面,可增强内皮细胞黏附和贴壁生长。混合预衬比单独预衬处理在增强细胞黏附性及贴壁生长方面效果更好。
目的：从犬骨髓细胞中分离出骨髓间充质干细胞,提取内皮祖细胞,体外培养分化为内皮细胞,种植到混合预衬去细胞牛颈静脉材料上培养,体外构建组织工程血管。
方法：1.使用Ficoll淋巴细胞分离液,通过密度梯度离心法从犬骨髓细胞中分离出单个核细胞,骨髓单个核细胞通过反复贴壁培养法获得内皮祖细胞,CD34抗原染色鉴定；含多种细胞生长因子的内皮细胞培养基培养,促进内皮祖细胞分化为成熟的内皮细胞,CD31和人类Ⅷ因子相关抗原染色鉴定。
2.混合预衬材料处理牛颈静脉脱细胞血管材料,将内皮细胞种植到血管材料,体外静态培养7天后,HE染色观察表明细胞覆盖情况及扫描电镜检测血管片上细胞的生长情况。
结果：原代培养的骨髓间充质干细胞呈螺旋状、放射状生长排列。原代培养的骨髓内皮祖细胞很快形成克隆,第2代细胞中CD34相关抗原的表达阳性率约45%,CD31和Ⅷ因子表现阴性。从第4代细胞起表现出内皮细胞样形态,呈“铺路石”状,并表现接触抑制的特点,第5代细胞CD34相关抗原表达阴性,CD31和Ⅷ因子相关抗原表达的阳性率为78%左右。种植于组织血管表面细胞,HE染色显示细胞覆盖血管表面,扫描电镜检测到血管片上均有较致密的细胞覆盖生长,但细胞排列无序。
结论：骨髓来源的间充质干细胞分化为内皮细胞,可种植于脱细胞牛颈静脉血管,构建内皮化组织工程血管。
目的：将种植内皮细胞的组织工程血管,用于重建犬右心室流出道实验模型,观察体内组织工程血管与血液接触的条件下的表面内皮化、细胞浸润以及钙化、血栓情况,评价内皮化去细胞牛颈静脉临床应用可行性。
方法：以种植分化扩增所获内皮细胞的去细胞牛颈静脉带瓣管道为研究对象,建立犬重建右心室—肺动脉连接的动物模型,设立未种植细胞的去细胞结合光氧化制备的牛颈静脉血管为对照组。实验动物分别于1月、3月和6月屠宰后取出标本,大体观察了解血管形态改变；组织化学和免疫组织化学染色观察各种炎性细胞浸润、表面内皮细胞生长,电镜检查了解植入后的管壁超微结构变化。
结果：17只犬除了5只死于手术中室颤外,余12只得以存活,其中对照组和试验组各6头。于1月、3月和6月各宰杀4只,对照组和试验组各2头。大体解剖标本：1月、3月对照组和试验组均无血栓和钙化形成,；6月对照组犬存在血栓沉着和近端吻合口膜样增生,存在血管部分钙化点,试验组均无异常。Ⅷ因子染色显示：1、3、6月时,试验组标本都表现出染色阳性,对照组只有6月的8因子染色结果显示为阳性。von kossa钙盐染色对照组在6月见到明显钙化点,试验组未见钙化点。电镜结果显示：试验组在1月时血管表面细胞部分覆盖,对照组无细胞。3月试验组标本表面细胞铺连成簇,对照组部分覆盖,可见裸露血管。6月试验组可见铺连成片细胞,对照组仍有部分组织工程血管显露。
结论：犬骨髓间充质干细胞分化为内皮细胞后,种植预衬处理后去细胞牛颈静脉血管,在重建右室流出道体内实验中表现出良好生长活性、抗钙化能力和较好内皮化程度。
Objective:To explore Surface Pre-coating method of decellularized Bovine jugular vessel sheets,to increase endothelial cells' biocompatibility,attachment and growth on the tissue Engineering material.
Methods:Pick out fresh and suitable bovine jugular vein as the tissue Engineering material.Acellular bovine jugular vein matrix is made through a gentle multi-process decellularization protocols and cross-linked by dye-mediated photooxidation.Apply Fibronectin, CollagenⅣ,gelatin as 3 single Pre-coating groups, Fibronectin and gelatin, Fibronectin and Collagen IV and gelatin as 2 mixtured Pre-coating groups, PBS liquid as the compared group.Six different compositions were deposed onto surface of 54 BJV sheets with a dose of 30μl,and each sheet,which was cut out at 1.6 cm2,was implanted with human umbilical vein endothelial cells(CRL-2480) 5×105 on surface. Then culture the sheet under static condition for 7 days.Took the sample at 1st,4th,7th day,depart cells from sheet surface with enzyme and count numbers.Took samples for HE stain and took photos on microscope.The 7th day samples of the compared and the mixtured-Pre-coating groups had scanning and transmission electron microscope checks.By comparing each groups'effect on increasing cell's biocompatibility,growth and disposition, explore the best surface pre-coating method of decellularized BJV sheets,so as to get the best endothelialization of BJV sheet.
Results:(1)Cell number:In 1st day of culture course,Pre-coating groups'surface cells,which is departed with enzyme,were more than compared group's(p<0.005).There was no significant difference between pre-coating groups.In 4th to 7th day,there was a significant difference between the single and mixture Pre-coating groups(p<0.05),the mixture Pre-coating groups had more cells.There was no obvious difference between the 2 mixture pre-coating groups.(2)All samples had a section after paraffin imbedding, then had a HE stain and took photos on microscope.At 1st day,each groups'surface had a cell layer,spread closely,cellular nucleus could see an accumulation.At 4th day,the compared group's surface cells had a decoherence,while the pre-coating group gained a cell layer well.At 7th day, the compared and single pre-coating group were scarce of cells, fibre below was partly uncoated.The mixtured pre-coating group's surface cells had formed a integrity layer,linked with fibre below. (3)Took the 7th sample for a scanning electron microscope.On the compared group's surface, the cells were scarce and no conjunction linked between cells or cell and sheet. The mixtured Pre-coating group had more cells.Cellular skeleton was formed and linked with vessel sheet fibre. Among cells, there was fusiform-shaped axon as linkage,which was a tight connection of cells and fibre. (4)Transmission electron microscope could see vacant brand among surface cells and fibre below BJC sheet.
Conclusion:Decellularized BJV sheets are capable of growing endothelial cells.Under Fn, ColⅣ,gelatin single modification, the sheet gain an augment of cells' biocompatibility, attachment and growth. Mixtured Pre-coating is better than single pre-coating on increasing cells' attachment and growth.
Objective:Bone marrow mesenchymal stem cells (MSCs) and endothelial progenitor cells(EPC) were separated from dogs'bone marrow and then incubated for differentiation into endothelial cells. The endothelial cells were implanted on decellularized bovine jugular vessel in order to form tissue-engineering vessels.
Methods:1.Dogs'bone marrow mononuclear cells(BM-MNCs) were separated by gradient centrifugation on Ficoll (density 1.077g/ml) from bone marrow and EPCs were collected through adhearance cultivateding repeatedly.Cells were identified by CD34 dyeing,then incubated with DMEM and factorsfor to help EPCs differentiate to normal endothelial cells which were identified by CD31 and VIII factor dying.
2.The normal endothelial cells were planted on bovine jugular vein matrixs in static condition for 7 days.Cells'coverage and cultivation were tested with HE dying.The morphologic structure was observed with photographed by scanning electron microscope (SEM).
Results:The adherent BM-MNCs grew in radiative and spiral way and were proved to be EPCs 45% of which showed positive for CD34 and negative for CD31 and factorⅧ-related antigen..Primary culture EPCs formed clones quickly.The 4th generation cells'morphologic structure were like endothelial cells',and contact inhibition was observed.78% of the 5th generation cells were positive for CD31 and factorⅢ-related and negative for CD34 antigen.The cells planted on the conduits'surface proved to be well cultivated over the surface by HE dying.And the SEM showed that the coverage on the surface was compact without proper order.
MSCs were incubated for orientated differentiation into smooth muscle-like cells which showed positive for a-SMA.
Conclusions:Bone marrow MSCs and EPc can be differented into ECs in vitro respectively. ECs can be successfully implanted on the acellular scaffolds of bovine jugular vein to built tissue-engineering vessels.
Objective:The conduits with cell plantation were used to reconstruct dogs' right ventricle outflow tract.This study was to investigate the histological change of endothelialization BJVCs and evaluate the possibility of which as a tissue engineering scaffold in clinical application.
Methods:Decelluarizated valved BJVCs with cell plantaion were used to rebuilt dogs'right ventricle outflow tract.The control group were decelluarizated valved BJVCs without cell platntaion.The conduits were explanted and analyzed by HE examination, light microscopy, and electron microscopy in 1 month,3 months and 6 months after being implanted. Tissue contents of collagen, elastin and glycosaminoglycans were assayed respectively to the explanted tissues.
Results:17 dogs subjected implantation,5 died of ventricular fibrillation in operation and the other 12 ones survived.4 dogs sacrifaced in 1 month after operation, another 4 dogs sacrifaced in 6 month and the others sacrifaced in 1 year. In 1st and 3nd month,the test and control group had no thrombus or calcification in Macroscopic observation.In 6th month, thrombus and ndomembrance proliferation was observed near the anastomotic stoma in the control group,and part of the conduits were calcificated.The test group was normal.FactorⅧ-related dying were observed positive in all time point in test group,while positive in 6th month in control group. Von kossa calcium dying were positive in 6th month vessel of control group,while nagtive in test group conduit of all time point.The Scanning electron microscope showed that cells were covered part of the test groups'surface,and the controul group scarce in 1st month.In 3th month,the test group gained more coverage of cells,and the control group gained partly.In 6th month,cells on the test group vessels were contacted tightly and like a plaque,but the control group were partly covered and fibers below was obvious.
Conclusions:Pre-coating decelluarizated valved BJVCs with cell plantaion have growth potentiality and supporting endothelialization, with low-immunogenicity and low-calcification, which surpport the conduit as a tissue engineering scaffold for reconstructing the connection of pulmonary arteries with right ventricles.
引文
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