Development of a mouse model for evaluation of small diameter vascular grafts

BACKGROUND: It is estimated that 80,000 individuals are unable to undergo life or limb saving bypass surgery because of inadequate small caliber synthetic vascular grafts. The use of tissue engineering methods has been proposed as a potential means of creating improved vascular conduits. We have developed a severe combined immunodeficiency (SCID) mouse aortic interposition model for initial evaluation and screening of small diameter vascular conduits in vivo. METHODS: Fifteen small diameter vascular conduits, approximately 1 mm in diameter and 10 mm in length, were implanted as infrarenal aortic interposition grafts for 1 to 35 days. Eight grafts were constructed using a decellularized ovine arterial tissue as a scaffold. Seven grafts were constructed from silastic tubing. Four grafts were composed of Polyurethane, and two were made of expanded polytetrafluroethylene. To explore noninvasive means of evaluating patency, grafts were selectively imaged using ultrasound and micro-computed tomography. RESULTS: All grafts were patent immediately post-operatively and at time of sacrifice. All imaging modalities were able to visualize the grafts and confirm patency. All specimens were sent for histology to evaluate neotissue formation and to correlate radiographic morphology with histological morphology. CONCLUSIONS: The use of the SCID mouse model for initial evaluation of small caliber grafts is feasible and provides a cost effective rapid screening model with the added advantage of being able to use human cells in further studies.     

Improved retention of endothelial cells seeded on polyurethane small-diameter vascular grafts modified by a recombinant RGD-containing protein

Sponge-type small-diameter vascular grafts were fabricated from a medical-grade polyurethane, Pellethane 2363-80A, by utilization of a salt casting technique. The grafts were compliance matched with a storage modulus of 0.53 +/- 0.08 MPa. The luminal surface of grafts was modified with a thin layer ( approximately 40 micro m) of gelatin crosslinked by epoxide. Then a special Arg-Gly-Asp (RGD)-containing recombinant protein, named CBD-RGD (cellulose binding domain RGD-containing protein), was coated onto the gelatin layer. The platelet adhesion and activation on such a gelatin/CBD-RGD modified surface was significantly reduced. Human umbilical vein endothelial cells were seeded more efficiently onto the modified grafts. There was also a substantial reduction in the subsequent loss of cells from the graft surface following perfusion in vitro. The cell number retained on the modified graft was enhanced by three times after 1 h of perfusion, and by eight times after 3 h of perfusion (retention rate approximately 63%). The retention after 3 h of perfusion could be further increased to nearly 100% if the lined endothelium on gelatin/CBD-RGD modified graft was cultured for another week before perfusion. The modified surface was also shown to help canine external jugular vein endothelial cells to maintain the round cell morphology in vitro.     

In vitro and in vivo study of ion-implanted collagen for the substrate of small diameter artificial grafts

Ion implantation into the collagen-coated inner surface of the grafts was performed and evaluated in vitro and in vivo to develop small diameter artificial vascular grafts. He+ ion implanted collagen-coated grafts with a fluence of 1 x 1014 ions/cm2 inhibited platelet adhesion and demonstrated patency for 240 days in an animal study. The platelet adhesion test using platelet rich plasma (PRP) showed antithrombogenicity at the fluence of 1 x 1014 ions/cm2. Washed platelet adhesion test showed thrombus formation at the fluence of 1 x 1014 ions/cm2. The results suggested that plasma protein adsorption onto the ion-implanted collagen significantly improved performance of these synthetic grafts.     

In vitro laser recanalization of chronically occluded prosthetic grafts.

Polytetrafluoroethylene (PTFE) and Dacron grafts were implanted in canine femoral and carotid arteries using PTFE and Prolene suture, respectively. Arteries containing occluded grafts were explanted and laser recanalization was attempted in vitro. Laser recanalization was successful in 78% of PTFE grafts compared to 30% of Dacron grafts. Recanalization was complete (residual stenosis less than 5%) in opened PTFE grafts, whereas residual stenosis averaged 60% in recanalized Dacron grafts. PTFE graft/PTFE suture anastomotic tensile strength was unchanged after recanalization, while Dacron graft/Prolene suture anastomotic tensile strength decreased significantly. In addition, anastomotic bursting pressure was significantly higher for lased PTFE grafts with PTFE sutures (300 mg Hg) compared to lased Dacron grafts with Prolene sutures (70 mm Hg). Chronically occluded PTFE grafts with PTFE suture can be safely and effectively opened by laser recanalization. In contrast, attempted laser recanalization of Dacron grafts sutured with Prolene suture is seldom successful, significantly weakens the graft artery anastomosis, and should be avoided.     

A comparison of CORVITA and expanded polytetrafluoroethylene vascular grafts implanted in the abdominal aortas of dogs

The utility of CORVITA vascular grafts, composed of an inner layer of meshed polyurethane fibers and an outer layer of meshed Dacron reinforcement, for replacement of the abdominal aorta was assessed in a canine model and compared with expanded polytetrafluoroethylene (ePTFE) grafts. CORVITA or ePTFE vascular grafts were implanted and left in place for 3 or 6 months. After removal, they were inspected macroscopically and histologically. Microspectrophotometry was used to quantify smooth muscle cells (SMCs), elastin (EL), and collagen (CL) in the media of the native artery. The patency rate of the CORVITA grafts after 6 months was 100%, whereas that of the ePTFE grafts was only 50%. Moreover, stenoses were apparent in all of the ePTFE grafts, but in only 43% of the CORVITA grafts. The intimal thickness at the distal anastomosis was significantly greater at 3 months in the ePTFE grafts (P<0.01), and there were significantly more SMCs in the host arterial media at the proximal and distal anastomoses in these grafts. Thus, better long-term patency can be expected with CORVITA grafts than with ePTFE grafts. This conferred advantage is most likely attributable to the less pronounced intimal hyperplasia which results from the proliferation of SMCs in the media of the native artery.     

Evaluation of a polyepoxy compound fixed biological vascular prosthesis and an expanded polytetrafluoroethylene vascular graft

Antithrombogenicity is one essential requirement for the successful use of small caliber vascular prostheses. In this study, a polyepoxy compound fixed, heparinized 4 mm diameter Baxter Denaflex vascular graft was evaluated against a 4 mm diameter Gore-Tex expanded polytetrafluoroethylene (ePTFE) vascular graft in the canine model. In addition to the thromboresistant characteristic conferred by heparinization, the crosslinking agent allowed the Denaflex graft to retain the original color of the native artery. Six centimeter long graft segments were implanted into the carotid arteries bilaterally in 5 dogs. The patency rate at 3 months for the Denaflex graft was 100% (five out of five) whereas in the control ePTFE graft, it was 40% (two out of five). The explanted Denaflex grafts exhibited softness and flexibility, and their luminal surfaces maintained a white color like that before implantation. To the contrary, the patent ePTFE grafts felt hard, and red thrombi covered large portions on their inner surfaces. Under microscopic observation, neointima formation was limited to regions near the anastomotic sites for both types of grafts. This experiment showed that the Denaflex vascular graft has an excellent antithrombogenic property and has a compliance similar to native arteries.     

Cultivation of endothelial progenitor cells on fibrin matrix and layering on dacron/polytetrafluoroethylene vascular grafts

Completely biological tissue-engineered vascular graft is an upcoming substitute for damaged blood vessel, but its clinical use is currently limited due to poor mechanical strength. Therefore, at present, polymeric small-diameter vascular grafts lined with endothelial cells (ECs) to reduce graft thrombosis may be a more viable option. Successful construction of EC-seeded artificial grafts faces some challenges such as (i) retention of endothelial lining; and (ii) availability of differentiated autologous cells. Biomaterial surfaces that are modified by depositing extracellular matrix (ECM) components may stabilize cells in the lumen against forces of blood flow. Adult stem cells such as endothelial progenitor cells (EPCs) circulate in the blood and they usually attach to the exposed matrix at the injured blood vessel site. Depending on the signaling capabilities of ECM, cells may differentiate into ECs,, and if a similar composition of the matrix is provided in vitro, EPCs isolated from blood might get differentiated and thus autologous cells for tissue engineering may be obtained. In this in vitro study, ECM scaffold consisting of biomolecules such as fibrin, fibronectin, and gelatin along with growth factors is found to have supported differentiation of EPC into EC. Further, the ECM precoated on Dacron and polytetrafluoroethylene is found to have supported the formation of EC monolayer that synthesized nitric oxide, and resisted shear stress. Thus, biomimetic fibrin composite is found to be suitable not only to seed cells on currently available artificial grafts but also to obtain differentiated EC from EPC.     

CD34+细胞与单个核细胞对新型小口径人工血管早期抗血栓形成影响的比较

目的 比较纯化的CD34+细胞与未经筛选的单个核细胞(uMNCs)作为种子细胞在小口径组织工程血管早期的抗凝作用.方法 uMNCs与CD34+细胞分离自犬骨髓,将两种细胞分别在体外培养、传代,内皮细胞生长因子诱导分化,体外血小板黏附实验检测细胞的体外抗血小板黏附功能.将等量自体上述两种细胞分别种植在小口径人造血管腔内表面并替代一段颈动脉.分别在24h、72 h、1周后取出移植血管行苏木素-伊红(HE)染色和扫描电镜检查.结果 体外血小板黏附实验显示CD34+细胞抗血小板聚集作用显著高于uMNCs.植入体内后,移植uMNCs血管腔表面比种植CD3+细胞的有更多的血小板黏附和血栓形成.24 h、1周后CD34+细胞组血栓面积与移植的血管腔表面积之比小于uMNCs组,差异有统计学意义(P＜0.05).结论 CD34+作为种子细胞种植小口径人工血管后比未经筛选的uMNCs具有更好的早期抗血栓形成功能.

Abstract：

Objective To compare the antithrombogenic property of purified CD34 + cells with that of unfractioned mononuclear cells (uMNCs) after seeding on the small caliber man-made grafts. Methods uMNCs and CD34 + cells were isolated from the canine bone marrow. Platelet adhesion assay was performed to determine antiplatelet adhesion property of the cells in vitro. Equal number of both cells were seeded onto the luminal surface of small caliber man-made grafts and implanted to replace a segment of common carotid artery. At time intervals of 24 h, 72 h, and 1 week, the grafts were retreived, HE staining and scanning election microscopy (SEM) exam were performed. Results Platelet adhesion assay indicated that there was more significant platelet adhesion on uMNCs than that on CD34 + cells. SEM revealed that there was more significant platelet adhesion and thrombus formation on the luminal surface of the grafts seeded with uMNCs than those seeded with CD34 + cells 24 h after implantation. The ratio of thrombi area to the luminal surface area of grafts in CD34 + cells group at 24 h and 1 week was statistically lower than that of the uMNCs group (P ＜0. 05). Conclusion CD34 + cells exhibit better antithrombogenic ability after seeding onto the small caliber vessel grafts as compared with uMNCs. CD34 + cells may be preferable cell source for better improve the thromboresistance and patency of small caliber man-made vessel grafts for their future clinical application.     

大鼠小肠Cajal间质细胞的体外分离、培养及鉴定

目的：探讨大鼠小肠Cajal间质细胞（ICC）的原代分离、培养及鉴定方法。 方法：出生后5~10 d的SD乳大鼠,颈椎脱臼处死。无菌条件下取出小肠,在解剖显微镜下剥去小肠系膜、肠黏膜和黏膜下层,将小肠平滑肌层组织剪成小块后接种于含有干细胞因子的DMEM培养基中进行培养。倒置显微镜下连续观察组织块周围细胞的游出情况及细胞的形态,用酪氨酸蛋白激酶受体c-kit特异性抗体免疫荧光染色鉴定细胞类型。 结果：培养1周后,倒置显微镜下可见组织块周围长出细胞,呈梭形、三角形,有多个短突起;随着培养时间的延长,突起细长化并彼此相互连接形成网络。该类细胞c-kit抗体免疫荧光染色呈阳性。 结论：该研究成功建立了一套由大鼠小肠平滑肌组织块原代培养ICC的方法,为ICC的生物学特性及其与胃肠道动力障碍性疾病关系的研究奠定了基础。     

不同组织来源生物补片降解性能的比较研究

目的比较3种不同组织来源生物补片的体内外降解性能。方法采用Ⅰ型胶原酶对来源于基底膜(BM)与小肠黏膜下层(SIS)复合细胞外基质、单纯小肠黏膜下层、戊二醛化学交联心包(PC)三种生物补片进行体外降解试验,确定各材料的降解率;采用各材料修补大鼠腹壁肌部分层次缺损评估3种补片的降解和组织修复情况。结果体外降解试验中,酶溶液作用120 h后,非交联的BM/SIS复合补片和SIS补片完全降解,PC补片的降解率仅为4.3%±1.9%。体内降解试验中,术后2个月,大体观和组织病理切片染色结果证实BM/SIS复合补片完全降解,组织重塑,再生胶原有序;SIS补片完全降解,再生胶原有序性较差;术后12个月,PC补片未见明显降解,未见细胞长入补片中央区。结论虽然BM/SIS复合补片的体内外完全降解时间均较短,但其可以实现组织的良好快速重塑,提示BM/SIS复合补片是一种降解与再生同步的组织修复材料。     

Continuous monitoring of central venous oxygen saturation in neonates and small infants: in vitro evaluation of two different oximetry catheters

BACKGROUND: Accurate assessment and monitoring of the cardiocirculatory function are essential during major pediatric and pediatric cardiac surgery. Monitoring of the central venous oxygen saturation (ScvO(2)) may be a better indicator of tissue oxygenation and derangement of cellular oxygen utilization than the more commonly used vital parameters. Therefore, we compared oxygen saturation measurements with thin fiberoptic oximetry catheters and standard blood gas oximetry in an in vitro setting. METHODS: Two different size continuous fiberoptic oximetry catheters (2-4-F) were inserted in an extracorporeal circuit filled with human red blood cells in normal saline (haematocrit 30%, flow 600 ml.min(-1)). The results of fiberoptic oximetry were then compared with standard blood gas oximetry for a wide range of different oxygen saturations using linear regression. RESULTS: The oxygen saturations found ranged from 9% to 100%. The results of the two different fiberoptic oximetry catheters correlated significantly (r = 0.99, P < 0.0001) with standard blood gas oximetry. CONCLUSION: The results of fiberoptic oximetry are nearly identical with standard blood gas oximetry for a wide range of different oxygen saturations. Thin oximetry catheters can be inserted percutaneously even in neonates and small infants. The continuous monitoring of ScvO(2) may be beneficial, especially in patients who are in danger of developing low cardiac output or sudden cardiovascular collapse.