组织工程软骨生物反应器的设计

目的 根据流体力学原理优化反应器内腔结构,并设计一套组织工程软骨生物反应器.方法 采用计算机仿真的方法对反应器内腔流场进行仿真计算,通过流场分析确定内腔结构,最终构建一套完整的生物反应器系统.结果 确定了生物反应器的内腔结构,生物反应器由控制系统和细胞培养室两部分构成,能置于培养箱中对软骨细胞材料复合物进行动态培养.结论 反应器内腔的结构是合理的.整个生物反应器系统运行可靠.     

组织工程软骨生物反应器的设计

目的 根据流体力学原理优化反应器内腔结构,并设计一套组织工程软骨生物反应器.方法 采用计算机仿真的方法对反应器内腔流场进行仿真计算,通过流场分析确定内腔结构,最终构建一套完整的生物反应器系统.结果 确定了生物反应器的内腔结构,生物反应器由控制系统和细胞培养室两部分构成,能置于培养箱中对软骨细胞材料复合物进行动态培养.结论 反应器内腔的结构是合理的.整个生物反应器系统运行可靠.     

组织工程软骨实验生物反应器的设计

目的:设计一套计算机控制的组织工程软骨实验用灌流型生物反应器。方法:通过计算机控制时间及流量,利用蠕动泵、密闭的硅胶管、玻璃管、储液瓶等串联设计一套无菌的灌流型软骨组织工程用生物反应器。结果:生物反应器由控制系统和培养系统组成,运行良好,能置于培养箱中对软骨细胞材料复合物进行动态培养。结论:反应器设计合理。整个生物反应器系统运行可靠。     

反应器内构建组织工程血管平滑肌层的实验研究

目的 探讨生物反应器内构建组织工程血管的可行性.方法 于6个月龄犬颈总动脉血管获取平滑肌细胞,经体外培养扩增后接种于聚羟基乙酸(PGA)上,形成细胞-材料复合物,并将其置于生物反应器内培养.实验组模拟成年哺乳动物循环系统的参数予以动态力学刺激培养(搏动频率:75次/分;扩张量＜5%);对照组为静态培养,其余与实验组相同,分别于3与6周后取材检测.结果 生物反应器内培养的血管大体观察具有良好的弹性,管腔圆,色泽光亮;HE染色显示平滑肌纤维成分排列较规则,有层次感;弹力纤维染色显示弹力纤维成分较多而密;免疫组织化学检测显示为棕黄色层状排列的平滑肌纤维.对照组弹性欠佳,管腔塌陷,色泽暗淡;平滑肌纤维与弹力纤维成分较少且排列紊乱,层次感差.结论 应用生物反应器可构建具有良好结构的血管样结构的平滑肌层组织.     

应用生物反应器构建组织工程化血管的实验研究

目的探讨利用生物反应器体外构建具有完整结构的组织工程化血管的可行性。方法从6月龄毕格犬颈总动脉获取平滑肌细胞,颈外静脉获取内皮细胞,经体外培养扩增。然后,先将平滑肌细胞接种于聚羟基乙酸(PGA)上,形成细胞—材料复合物,将该复合物置于生物反应器内培养。模拟成年哺乳动物循环系统的参数,予以动态力学刺激(搏动频率:75次/分;扩张量<5%)。8周后,在其上接种内皮细胞,对照组不接种内皮细胞,培养5d后取材检测。结果组织学染色显示,平滑肌纤维成分排列较规则,有层次感,内皮细胞完整;对照组未见内皮细胞层结构。结论利用生物反应器可在体外构建具有完整结构的组织工程化血管。     

组织工程技术构建小口径血管的实验研究

目的 探讨运用组织工程技术构建血管组织的方法。方法 将体外培养、扩增的新生婴儿脐静脉血管内皮细胞与平滑肌细胞接种于片状聚羟基乙酸材料上,形成细胞-材料复合物,将该复合物包绕于硅胶管使成管状结构后移植于裸鼠皮下,第2及第6周后行大体及组织学检测。结果 组织工程技术构建的血管组织大体结构与天然血管相似,免疫组化显示组织工程化血管最内层有Ⅷ因子阳性细胞覆盖,管壁内有大量细胞外基质及散在的α-平滑肌肌动蛋白阳性细胞存在。结论 运用组织工程技术可形成具有与正常血管组织学结构相似的血管。     

应用脂肪干细胞构建组织工程化小口径血管平滑肌层的实验研究

目的探索利用脂肪干细胞在生物反应器内构建组织工程血管平滑肌层的可行性。方法用抽吸的脂肪获取脂肪干细胞,在生长因子TGF-β1和BMP4作用下诱导成平滑肌细胞,然后将诱导的平滑肌细胞接种于PGA上,将细胞-材料复合物置于生物反应器内进行培养,在模拟胚胎发育血流动力学的刺激下(搏动频率:75次/分;扩展量<5%),构建小口径的血管平滑肌组织。培养8周后,取材行组织学和生物力学检测并与正常血管对比。结果脂肪干细胞在TGF-β1和BMP4的诱导下,细胞呈现平滑肌细胞特有的"波峰-波谷"样生长特点,并表达平滑肌细胞的特异性标记物α-SMA、SM22α、calponin和SM-MHC;反应器内培养8周后,构建的管状组织胶原分泌旺盛,具有一定的力学强度和弹性。结论利用脂肪干细胞可在体外生物反应器内构建组织工程化小口径血管平滑肌层,为临床上小血管病变的修复提供了一种新的可能的途径。     

应用生物反应器体外构建组织工程化血管平滑肌层

目的探讨生物反应器内构建具有一定强度的组织工程化血管的可行性。方法将体外培养扩增的猪血管平滑肌细胞接种于聚羟基乙酸(PGA)上,形成细胞材料复合物,将其置于生物反应器内培养。实验组(n=5)为动态力学刺激培养(搏动频率:75次/分;扩张量:<5%);对照组(n=5)为静态培养。3周后行大体观察及组织学检测。结果血管样组织直径5mm,长10mm,厚1mm。实验组具有良好的弹性,管腔圆;平滑肌纤维与弹力纤维较多,排列有层次。对照组弹性欠佳,管腔塌陷;平滑肌纤维与弹力纤维较少且排列紊乱。结论应用生物反应器可构建具有良好弹性的血管平滑肌层组织。     

利用ADSCs在生物反应器中构建小口径血管的初步研究

目的 尝试利用人的脂肪基质干细胞(Adipose-derived stromal cell,ADSCs)在生物反应器中构建组织工程化小血管.方法 采用脂肪抽吸术获取人脂肪组织,将脂肪细胞分离、传代培养;对培养的第三代细胞进行脂肪干细胞表型鉴定;同时称取一定量的聚羟基乙酸(PGA)制成片状材料,消毒晾干备用,将细胞接种在PGA材料上,静态培养半天后置于生物反应器中动态培养4周,然后进行初步检测.结果 从形态学观察,细胞伸展呈成纤维细胞样,生长有方向性,5～6 d后可见克隆开始形成.对第三代ADSCs进行检测,结果显示除CD166、CD105有高的表达外,CD14、CD34表达很低.从反应器取出的样品能形成血管样组织,扫描电镜观察:样品基质分泌旺盛.结论 采用组织工程方法,从动态培养的角度可以在体外利用ADSCs构建血管形状的组织,为进一步研究ADSCs在血管组织工程中的应用提供了一种好的方法.     

组织工程化肌腱体外构建的环境优化及系统设计

目的 设计一套构建具有一定功能和形态的肌腱组织的体外培养方法。方法 根据肌腱细胞体内的生物力学环境，在细胞的培养过程中，设计一套能够提供张力和参数测量系统的生物反应器，在肌腱的培养过程中对肌腱施加不同形式的张应力作用，以期在体外获得力学性能优化的自体肌腱。结果 细胞培养和张应力控制系统是由完全透明的有机材料制成的，整个系统由细胞培养室、测量系统和排换液系统等部分构成，在长期的肌腱细胞培养中，可提供肌腱一材料支架动态的培养方式。细胞培养室能很方便与气动肌腱连接，产生持续的脉冲张力作用。结论 用肌腱生物反应器可以使肌腱组织复合物在形成过程中始终处于力学环境中，促进肌腱组织中胶原纤维的平行排列，提高肌腱的力学性能，为组织工程化肌腱的产业化提供一种新的方法。     

利用脱细胞基质预构小口径组织工程血管的实验研究

目的 在犬腹腔内预置脱细胞基质后形成结缔组织管,观察将其作为血管替代物的力学特征及移植于自体股动脉后的组织结构变化.方法 在犬腹腔内埋置长8～12 cm用硅胶棒支撑的制备好的脱细胞基质,3周后将硅胶棒周围形成的管状物作为血管替代物移植于自体股动脉,同时对此管状物作力学、组织学检测及与颈动脉、股静脉的对比分析.在移植术后6个月观察血管通畅情况.光镜、透射及扫描电镜观察替代物的组织结构.结果 ①血管替代物的力学性能弱于正常动脉而强于正常静脉.②组织学观察:形成的管状物内腔有少量的间皮细胞黏附;弹性纤维、胶原纤维结构较完整;纤维网中充满成纤维母细胞.6个月后内皮细胞在替代物内壁覆盖连续,平滑肌细胞与对照组相近.③移植6个月后脱细胞基质组血管全部通畅.结论 ①用脱细胞基质预构的血管替代物力学性能符合血管移植、血运重建的需要.②所形成的血管替代物移植6个月后组织相容性良好,替代物管壁厚度及组织结构已接近正常血管.     

一种新型血管生物反应器的研制

目的:研制适合于组织工程血管体外构建的生物反应器。方法:采用MTT法对聚氨基丙烯酸甲酯(PMMA)、乳胶、硅胶三种材质的浸提液行细胞毒性试验;构筑反应器的三大部分;确定各项搏动参数。结果:PMMA、硅胶材质无细胞毒性,适合于实验所需。达到5%的搏动幅度和75～120次/mi n的频率。结论:该反应器可用于组织工程血管、心瓣膜等器官的体外构建。     

Experimental vascular graft using small-caliber fascia-wrapped fibrocollagenous tube: short-term evaluation

This study was undertaken to evaluate the application of autogenous fascia as a framework for a fibrocollagen tube for small-caliber vascular prostheses in Japanese white rabbits (n = 15). The fascia, measuring 10 x 40 mm, was harvested from the dorsal fascia and was carefully wrapped around a silicone rod of 1.5 mm in diameter. Then, the fascia-wrapped silicone rod was implanted into a subcutaneous pocket on the medial thigh. Four weeks later, the fascia-wrapped silicone rod was removed from the subcutaneous pocket. The rod was removed from the material, and the fascia-wrapped fibrocollagen tube was treated to make it antithrombogenic. Subsequently, the 1.5 x 10-mm fascia-wrapped tube was prepared as an arterial conduit. Using microvascular techniques, the tube was interposed into the divided femoral artery. Eleven of 15 grafts maintained patency over the follow-up period and no aneurysmal formation was found at any graft site. Microscopically, there was an ingrowth of endothelium with fibroblast proliferation from each end of the recipient vessel at 2 weeks after interposition; however, no neointima was found to line the center of the conduit. At 5 weeks after interposition, the neointimal growth rate was 70 percent. There was an ingrowth of endothelium with fibroblast proliferation from each end onto the entire internal surface of the conduit at 8 weeks after interposition. Further study is required before any long-term conclusions can be drawn.     

Microsilicone anastomoses

Silicone tubes used for lacrimal system reconstruction have already been tested as temporary microvascular shunts. As a model of the small vessel, the silicone tube is used for initial training in microvascular surgery. It is useful for both teaching and practicing.     

A new intracranial Silastic encircling clip for hemostasis. Technical note

A new encircling clip made of a silicone tube has been designed for treating accidentally injured cerebral vessels. No special holders are necessary. This clip can be tailored depending on the shape of the injured vessel. The clip is a simple and effective tool for achieving complete hemostasis.     

The use of immobilized protamine in removing heparin and preventing protamine-induced complications during extracorporeal blood circulation

Heparin, currently used in extracorporeal blood circulation procedures, may lead to hemorrhagic complications. Protamine, used for reversal of heparin-induced anticoagulation at the end of such procedures, can cause adverse hemodynamic responses. To prevent both types of complications, we have developed a reactor device containing immobilized protamine (i.e., a protamine bio-reactor) that can be placed at the distal end of the circuit, thus providing simultaneous extracorporeal heparin removal and protamine treatment. In preliminary in vivo studies involving dogs at a blood flow of 100 ml/min, the bio-reactor removed about 50% of the administered dose of heparin (i.e., 100 units/kg) in 10 min. While rapid injection of protamine in dogs anticoagulated with heparin produced a transient and significant (P less than 0.005) decreases in systemic arterial blood pressure (-39.5 +/- 9.2 mmHg), cardiac output (-1.59 +/- 0.23 L/min), and mixed venous oxygen saturation (-7.5 +/- 1.3%) and increases in pulmonary artery systolic (+12.7 +/- 4.4 mmHg) and diastolic pressures (+10.0 +/- 3.6 mmHg), the use of the protamine bio-reactor did not elicit any statistically significant change in any of the variables measured. Hemolysis was not significant, as reflected by a statistically insignificant change of the animals' red blood cell counts, hematocrits, and total hemoglobin values. In addition, hemolytic complement was found to be reduced only by 10% in animals with the protamine bio-reactor, whereas it was reduced rapidly by 20% in animals receiving intravenous protamine administration and progressively by 20% in control animals with a sham reactor that contained no protamine. Furthermore, the use of the protamine bio-reactor also significantly reduced the protamine-induced transient thrombocytopenic and granulocytopenic responses. The white blood cell counts and platelet counts decreased to 87.7 +/- 7.5 and 83.3 +/- 5.0% of baseline, respectively, in dogs with the protamine bio-reactor compared to 35.5 +/- 14.3 and 32.1 +/- 8.1% of baseline in dogs receiving intravenous protamine. The protamine bio-reactor may provide a unique means to simultaneously control both heparin- and protamine-induced complications.     

Circumferential tracheal replacement with costal cartilage

Long-term incorporation of foreign material or tissue in circumferential tracheal replacement will lead to progressive growth of granulation tissue, provoking either airway stenosis or a reduction of subepithelial blood supply and thereby preventing the appearance of functioning ciliated epithelium in the replaced segment. In experience with dogs, a 5 cm length of the thoracic trachea was replaced circumferentially with fresh autogenous untreated circularly positioned cartilage-perichrondrium strips. During the period of strengthening of this neotracheal wall, a temporary tracheal prosthesis made of tubular silicone rubber with absorbable sewing rings of polyglactin 910 placed 3 mm from the end of the tube served as a tracheal replacement. Six months after the cartilage transplantation, the neotracheal wall had stabilized sufficiently for the silicone rubber tube to be extracted with an endoscope. Four weeks after extraction of the silicone rubber tube the neotracheal segment was completely covered with ciliated epithelium over a thin subepithelial, well-vascularized layer. Subepithelial vessels had a diameter of 180 microm. They were a continuation of the intercartilaginous main vessels of the neotracheal wall. The presence of normal cilia on the epithelium was proved through transmission electron microscopy. Even the tubules of the cilia were arranged in the right order. In the ink test, movement of the transport marker across the neotracheal segment at a speed of 18 to 21 mm/min was proof of mucociliary clearance. In a process of migration starting from the margin of the trachea, the ciliated epithelium pervaded and replaced the preexisting temporary one-layer stratified squamous epithelium. This completely new technique of circumferential tracheal replacement with autogenous cartilage, avoiding permanent incorporation of foreign material, succeeds within the observation period of up to 7 months.     

An in vitro cell culture system to study the influence of external pneumatic compression on endothelial function

PURPOSE: External pneumatic compression (EPC) is an effective means of prophylaxis against deep venous thrombosis. However, its mechanism remains poorly understood. Understanding of the biological consequences of EPC is an important goal for optimizing performance of the EPC-generating device and providing guidance for clinical use. We present a new in vitro cell culture system (Venous Flow Simulator) that simulates blood flow and vessel collapse conditions during EPC, and we examine the influence of these factors on endothelial cell (EC) fibrinolytic activity and vasomotor function. METHODS: An in vitro cell culture system was designed to replicate the hemodynamic shear stress and vessel wall strain associated with induced blood flow during different modes of EPC. Human umbilical vein endothelial cells were cultured in the system and subjected to intermittent flow, vessel collapse, or a combination of the two. The biologic response was assessed through changes in EC morphology and the expression of fibrinolytic factors tissue plasminogen activator, plasminogen activator inhibitor type 1, profibrinolytic receptor (annexin II), and vasomotor factors endothelial nitric oxide synthase and endothelin-1. RESULTS: The cells remained attached and viable after being subjected to intermittent pulsatile flow (F) and tube compression (C). In F and F + C, cells aligned in the direction of flow after 6 hours. Northern blot analysis of messenger RNA shows that there is an upregulation of tissue plasminogen activator expression (1.95 +/- 0.19 in F and 2.45 +/- 0.46 in FC) and endothelial nitric oxide synthase expression (2.08 +/- 0.25 in F and 2.11 +/- 0.21 in FC). Plasminogen activator inhibitor type 1, annexin II, and endothelin 1 show no significant change under any experimental conditions. The results also show that pulsatile flow, more than vessel compression, influences EC morphology and function. CONCLUSION: Effects on ECs of intermittent flow and vessel collapse, either individually or simultaneously, were simulated with an in vitro system of new design. Initial results show that intermittent flow associated with EPC upregulates EC fibrinolytic potential and influences factors altering vasomotor tone. The system will facilitate future studies of EC function during EPC.     

体外组织工程血管支架内皮化的实验研究

目的：研究兔血管内皮细胞种植于组织工程血管支架内腔面的生长状况。方法：（1）将聚羟基乙酸（Plyglycolic acid,PGA)纤维无纺网和胶原纤维相混合，设计构建组织工程血管支架材料。（2）采用酶消化法从兔主动脉中分离培养兔血管内皮细胞并传代，纯化，接种于组织工程血管支架的内腔面，体外培养，并行电镜等观察。结果：该支架具有一定弹性和韧性，内皮细胞在其内表面形成较完整内皮细胞层，生长状况良好。结论：胶原包埋处理的PGA支架可以作为组织工程人工血管研究的较理想支架材料。为组织工程方法构筑具有分层结构的组织工程血管打下实验基础。