组织工程化人工神经实验研究

目的 研究组织工程化人工神经修复大鼠2.5cm长坐骨神经缺损的效果。方法 21只2月龄Lewis lw雌性大鼠随机分成三个神经移植组,每组7只。A组:种植同源雪旺细胞并具有内部支架结构的胶原神经管,即组织工程化人工神经。B组:无雪旺细胞但具有内部支架结构的胶原神经管。C组:自体神经移植组。术后六个月,进行系列神经电生理监测,神经肌肉组织学观察,S-100和神经微丝蛋白(Neurofilament)免疫组化染色,轴突计数等检查。结果 在A组和C组移植神经上均能诱发出波幅明显的神经肌肉复合动作电位(CMAP),再生轴突已通过移植神经全长,远端肌肉轻度萎缩。而B组中没有或仅记录到极小波幅的CMAP,移植神经远端结缔纤维组织增生,再生轴突罕见,所支配肌肉明显萎缩。结论 初步结果显示:组织工程化人工神经可用来修复大鼠长段神经缺损。     

Development of a model system for preliminary evaluation of tissue-engineered vascular conduits

Background/Purpose

The ability to construct tissue-engineered neovessels for use as arterial or venous grafts holds great promise for the advancement of pediatric surgical disciplines. Although the feasibility of tissue engineering vascular grafts has been demonstrated, the long-term function, safety, and efficacy of these grafts as well as their capacity to grow and adapt remain largely unknown. In an attempt to further characterize and develop this technology, we used severe combined immunodeficiency beige (SCID/bg) mouse recipients, chosen because such animals accept xenogenic human cells, to create a small animal model that would allow a rapid and cost-effective preliminary evaluation of the function of tissue-engineered vascular grafts.

Methods

Eight CB-17 SCID/bg female mice underwent vascular graft placement. Four of these mice received aortic interposition grafts, 1 mouse received an inferior vena cava interposition graft, and 3 mice underwent aortocaval graft insertion. All grafts were fashioned from decellularized ovine arteriole tissue engineering scaffolds. Grafts were evaluated for patency using clinical examination, ultrasound interrogation, and micro-computed tomography. Animals were killed at various time points after implantation, and grafts were harvested and analyzed histologically using standard hematoxylin and eosin staining.

Results

All grafts were patent based on clinical examination for up to 35 days. Patency was confirmed in 5 grafts using ultrasound interrogation. Patency was confirmed in 4 grafts using micro-computed tomography. One animal that underwent arteriovenous grafting had to be euthanized secondary to high-output cardiac failure on postoperative day 2. The remaining animals were killed between postoperative days 12 and 35. Histologic evaluation of the specimens demonstrated patent grafts with cellular ingrowth into the tissue engineering scaffold.

Conclusions

From these results, we conclude that the use of the SCID/bg mouse model for preliminary evaluation of new tissue engineering methodologies for construction of vascular conduits is feasible. Use of this model has the added advantage of evaluating nonautologous and even xenograft tissues, including human cells.     

骨软骨复合体修复软骨及软骨下骨缺损

目的探讨以诱导的兔骨髓基质细胞与双层PLGA支架构建组织工程骨软骨复合体,修复兔膝关节软骨及软骨下骨缺损的方法及结果。方法健康新西兰兔28只,分为三组。A组为常规培养MSCs(10只),B组为诱导培养MSCs(10只),C组为自体骨软骨块移植(8只)。密度梯度离心法获得骨髓基质干细胞(marrow-derivedstromalcells,MSCs),分别使用常规培养液和成软骨诱导条件培养液进行体外扩增传代。提取MSCs及关节软骨细胞总RNA,RT-PCR检测Ⅰ、Ⅱ型胶原表达。扫描电镜观察MSCs在PLGA双层支架上的复合与分布情况。A、B组MSCs分别与PLGA双层支架构建直径3.5mm、高3.0mm的骨软骨复合体,植入股骨髁髌骨滑车同比例骨软骨缺损区,术后第4、8、16、24周取材,进行大体观察、组织学检查和评分。结果RT-PCR见常规培养MSCs表达Ⅰ型胶原,无Ⅱ型胶原表达;诱导后MSCs表达Ⅰ、Ⅱ型胶原。扫描电镜观察MSCs在PLGA支架黏附生长良好,孔隙深处可见细胞分布。B组标本术后24周大体观察与正常软骨无明显差别,组织学检查为成熟的类透明软骨组织(4/6),优于A组(1/4)。结论MSCs具有成骨和成软骨潜能,可在基于细胞的软骨修复中作为种子细胞与双层PLGA构建组织工程骨软骨复合体,该复合体在实验动物模型中可修复关节软骨和软骨下骨缺损。     

组织工程组织血管化研究新进展

组织工程的血管化问题是制约组织工程产品的关键问题之一。根据血管发生中内皮细胞的来源不同，将血管发生分血管生成和血管再生两种形式，两种血管形成的方式都是一个动态、复杂的生理过程，通过生长因子和粘附物质来调节。对组织工程组织的血管化研究模型有体外和体内两种。体外实验可以检测支架与内皮细胞的粘附、增殖及迁移、材料的细胞毒、血管化效应和各种生长因子的调节作用，体内最简单实用的模型是用鸡胚茸毛尿囊膜模型，更高等的实验模型是在完整的动物体。组织工程组织血管化的策略有对支架材料的表面结构进行修饰、在材料内复合缓释的生长因子、内皮细胞与其它种子细胞联合培养、体内血管网包裹、血管束植入、血管模板以及体外在人造组织内构造微血管等，但目前的方法或多或少地存在一些问题，相信随着对血管化的机制和分子基础的了解的不断深入，以及各种技术手段的不断发展、完善，最终会找到比较理想的血管化方法。     

小口径猪血纤维蛋白血管支架内皮化的研究

明体外培养7 d内皮细胞在血管支架内表面就已形成较完整的内皮细胞单层,内皮细胞覆盖率达(99.2±0.5)%(n=4).结论 犬静脉内皮细胞在小口径血管支架内表面能较好地生长增殖.     

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.     

血管束、感觉神经束植入组织工程骨修复大段骨缺损的成骨研究

目的 观察血管束、感觉神经柬植入组织工程骨修复兔股骨大段骨缺损的成骨特点,探讨其对骨修复的影响. 方法 36只新西兰大白兔均制备左侧股骨干1.5 cm节段性骨缺损模型,随机分为三组(n=12),组织工程骨组(A组):自体骨髓基质干细胞复合β-磷酸三钙构建组织工程骨植入骨缺损;血管束植入组(B组):组织工程骨与血管束同时植入骨缺损;感觉神经束植入组(C组):组织工程骨与感觉神经束同时植入骨缺损.各组动物术后1、3、6个月行X线检查及影像学评分,同时每组各处死4只动物行大体及组织学观察.结果 影像学评分显示各时间点B组与C组的成骨优于A组,差异有统计学意义(P<0.05);B、C组间成骨差异无统计学意义(P>0.05).组织学观察显示新生骨多出现在血管周围,成骨方式以软骨内成骨为主. 结论血管束、感觉神经束植入组织工程骨的方法能更好地促进组织工程骨成骨及大段骨缺损修复.     

Tissue-engineered cartilage using fibrin/hyaluronan composite gel and its in vivo implantation

The importance of scaffold biomaterials has been emphasized for in vitro culture of tissue-engineered cartilage in a three-dimensional (3D) environment. In this study, we examined the feasibility of fibrin glue, mixed with hyaluronic acid (HA) as a composite scaffold. Fibrin glue has been a useful cell delivery matrix for cartilage tissue engineering and HA is a key component of normal articular cartilage. Our hypothesis is that compared to fibrin itself, a fibrin/HA composite can have significantly enhanced properties, due mainly to the added benefits of HA in the matrix. Pieces of cartilage were isolated from rabbit knees and the chondrocytes were harvested through enzymatic digestion. Both fibrin and fibrin/HA composite were prepared and subsequently implanted in nude mice (n = 9, each group) for 1, 2, and 4 weeks, respectively. The retrieved specimens were then analyzed and the results were compared. Cartilage-like tissue formation was detected earlier with fibrin/HA specimens. They produced significantly higher amounts of the extracellular matrix (ECM) molecules, GAG, and collagen at each time point than those in fibrin. Interestingly, the fibrin/HA composite was also competent in maintaining its initial size. Histology--Safranin O/fast green and Alcian blue--of the retrieved specimens found more intense, uniform staining in the fibrin/HA composites. Analysis of the gene expression of the ECM molecules also confirmed the benefits of the composite with added HA in the maintenance of phenotypic stability. The present study suggests that fibrin/HA composite may serve as a dependable cell delivery vehicle as well as a structural basis for tissue-engineered cartilage.     

A hybrid compliant vascular graft seeded with microvascular endothelial cells extracted from human omentum

We report the development of a hybrid vascular graft using an innovative compliant poly(carbonate-urea)urethane unlike any previous polyurethane MyoLink as a permanent scaffold. The engineered graft has a hierarchical arterial structure: a monolayer of oriented microvascular endothelial cells (MVECs) and 3-D matrix (human collagen Type 4/dermatan sulfate) bonded onto MyoLink. The grafts' clinical feasibility was evaluated by determining optimal MVEC seeding density, incubation time, viability, and adhesion of these cells when exposed to arterial shear stress. MVECs from human omentum were isolated by a new centrifugation protocol, radiolabeled and seeded onto hybrid graft 2 to 18 x 105 cells/cm(2) for 24 h at 37 degrees C and for 1 to 5 h at 6 x 105 cells/cm(2), washed 3 times, and gamma counted. Qualitative assessment of seeding density/incubation was also undertaken with scanning electron microscopy (SEM) and viability tested with a modified Alamar Blue assay. A pulsatile flow phantom was used to subject the hybrid graft (200 mm length, 5 mm internal diameter) seeded with radiolabeled MVECs (6 x 105 cells/cm(2) at 2 h) to arterial shear and dynamic scintigraphy images acquired in real time using a nuclear medicine gamma camera system during 14 h of perfusion (n = 6). The optimal seeding density was 6 x 105 cells/cm(2), and qualitative SEM confirmed this. Incubating cells for 2 h produced significantly greater cell attachment than was seen for 1 h incubation (p < 0.05), and there was no significant difference in adhesion between cells incubated over the 2 h. Exposure of grafts to acute shear stress resulted in significantly higher proportions of initial cells attached to hybrid grafts compared to native MyoLink grafts (67 +/- 3% versus 55 +/- 2%, p < 0.001). As shown here, tissue engineering of native Myolink grafts significantly reduces the seeding density and incubation time to produce a monolayer onto which cells adhere to better.     

A novel strategy to engineer small-diameter vascular grafts from marrow-derived mesenchymal stem cells

Tissue-engineered blood vessels have mainly relied on endothelial cells (ECs), smooth muscle cells (SMCs), and biocompatible materials. However, long-term results have revealed several material-related failures, such as stenosis, thromboembolization, and the risk of infection. Furthermore, SMCs from elderly persons have reduced capacity in proliferation and collagen production. Mesenchymal stem cells (MSCs) have the ability to differentiate into multiple cell lineages, including osteoblasts, chondrocytes, ECs, and SMCs. In the current experiment, rabbit MSCs were cultured to form a cell sheet. A tissue-engineered vascular graft (TEVG) was fabricated by rolling the MSC sheet around a mandrel. The TEVG was implanted into a defect of the common carotid artery after it was examined macroscopically and microscopically. Hematoxylin and eosin staining showed that cell sheet was composed of five to seven layers of cells with the thickness of 40-50 μm. Results from the adhesion assay revealed that MSCs had similar antiplatelet adhesion property to ECs. Histological analysis of TEVGs showed that the layers of the cell sheet had fully fused in vitro. After implantation, TEVGs had excellent patency and integrated well with the native vessel. The structure of the TEVGs was similar to that of the native artery 4 weeks after implantation. Electron microscopy showed that the implanted TEVGs endothelialized. These results indicated that a completely biological TEVG could be assembled with autologous MSCs. These TEVGs are useful for revascularization in humans, which would reduce the occurrence of complications caused by foreign materials.     

股静脉作为血管移植材料可行性与安全性的应用解剖研究

目的探讨股静脉作为血管移植材料的可行性与安全性。方法取60具成人尸体共114侧下肢标本,解剖观察股静脉、股深静脉、腘静脉及静脉交通支,测量股深静脉汇入股静脉处至收肌腱裂孔下缘的股静脉段长度,即股静脉可切取的解剖长度,以及静脉压扁外径。分析2010年3月-2011年5月收治的120例下肢股静脉段血栓形成患者CT静脉造影(computed tomography venography,CTV)检查资料,观察其下肢静脉回流通路。结果男性尸体身高平均158.3 cm,股静脉可切取长度为(18.8±2.3)cm,相对长度为0.118±0.013,静脉压扁外径为(15.8±0.8)mm;女性尸体分别为149.2 cm、(15.1±1.5)cm、0.101±0.010、(14.0±1.1)mm。男女股静脉可切取长度比较,差异有统计学意义(t=6.354,P=0.000);静脉压扁外径比较差异有统计学意义(t=5.555,P=0.000)。股静脉可切取长度与身高成正相关(r=0.964,P=0.000),股静脉压扁外径与身高成正相关(r=0.382,P=0.003)。解剖观察见16侧(14.0%)肢体存在双股静脉变异支,48侧(42.1%)肢体存在1支股-腘静脉交通支,38侧(33.3%)肢体存在1支股深-腘静脉交通支。CTV检查示,下肢股静脉血栓形成以后,大隐静脉及股-腘或股深-腘静脉交通支可代偿股静脉。结论大隐静脉和股-腘静脉交通支或股深-腘静脉交通支的存在,保证了切取股静脉作为血管移植材料的可靠性及安全性。     

组织工程血管研究内容及性能评价

心血管疾病已成为人类社会的多发病,每年有许多患者需要接受血管移植手术.组织工程血管的出现和应用,特别是纳米材料的应用,将有希望解决血管来源问题.本文就组织工程血管的研究内容及其性能评价作一综述.     

血管移植物置换犬颈总动脉的研究

目的 观察以猪血纤维蛋白/微孔聚氨酯弹性膜为管形支架内皮化构建的小口径血管移植物在体内血流动力条件下血管壁重塑过程.方法 用体外培养的小口径血管移植物置换6条犬双侧颈总动脉,于术后1 d、1周、2周、4周行影像学检查,并于术后5 d、2周、4周取材行组织学、免疫组织化学和扫描电镜检查以评价移植血管在体内重塑.结果 10根血管移植物中有8根仍保持通畅(通畅率80%);8根通畅的血管在术后至4周的不同时点取出发现其内表面菲薄、光滑,被覆盖一连续、鹅卵石样单层细胞,Ⅷ因子相关抗原抗体染色阳性.术后4周时新生动脉壁厚900μm,并于血管壁中层可见较多平滑肌细胞,而且最早于术后4周时在血管壁中层就可见弹力纤维.结论 猪血纤维蛋白/微孔聚氨酯弹性膜管形支架内皮化体外构建的小口径血管移植物在体内重塑后,可形成具有类似自体动脉壁结构.     

经诱导的骨髓基质干细胞应用于组织工程化人工神经的实验研究

目的 探讨骨髓基质干细胞应用于组织工程化人工神经修复大鼠10mm长坐骨神经缺损的效果。方法 28只体重在160～200g的雌性F344大鼠随机分成4组，每组7只。A组：种植经诱导5d后的同源骨髓基质干细胞并具有内部支架结构的中空管；B组：种植同源许旺细胞并具有内部支架结构的中空管；C组：无细胞只具有内部支架结构的中空管；D组：自体神经移植组。术后3个月，进行系列神经电生理监测、坐骨神经功能指数测定、神经组织学观察、S—100及神经微丝蛋白兔疫组化染色和轴突计数等检查。结果 术后12周内，实验组(A组)的各项检测指标均优于C组(P＜0．05或0．01)，与B和D组间差异无显著性(P＞0．05)。结论 初步结果显示经诱导的骨髓基质干细胞可作为外周神经组织工程中的种子细胞，并应用于人工神经修复外周神经缺损。     

纤维蛋白胶联合转染金属蛋白酶组织抑制剂-1预防自体移植静脉再狭窄

目的 为减少冠状动脉旁路移植术后移植静脉再狭窄,探讨术中应用纤维蛋白胶联合转染金属蛋白酶组织抑制剂-1(TIMP-1)对自体移植静脉再狭窄的影响及其作用机制.方法 将24只新西兰大耳白兔随机分为3组,每组8只,即非支架组(NS组)、纤维蛋白胶外支架组(FS组)、纤维蛋白胶联合转染TIMP-1组(TS/FS组) 建立兔颈外静脉颈总动脉旁路移植术模型.术后28 d取出移植静脉桥,进行组织病理分析移植静脉内膜厚度、中膜厚度及内膜面积、中膜面积,用逆转录-聚合酶链反应(RT-PCR)和Western blot法检测各组移植静脉中TIMP-1基因的表达.结果 术后28d,TS/FS组移植静脉内膜厚度及内膜面积分别为(37.98 ±4.19) μm及(0.557±0.049) mm2,与NS组的( 76.87±4.32) μm、(1.025 ±0.103)mm2及FS组的(50.28 ±4.69) μm、(0.743±0.052) mm2比较,明显减少(P＜0.05);TS/FS组移植静脉中膜厚度及中膜面积分别为(28.45 ±3.01) μm及(0.458 ±0.053) mm2,与NS组的(55.98±4.33) μm、(0.944±0.084) mm2及FS组的(36.46±4.36) μm、(0.643 ±0.056)mm2比较,差异有统计学意义(P＜0.05).与NS组和FS组比较,TS/FS组TIMP-1基因的mRNA和蛋白表达明显增强,差异有统计学意义(P＜0.05).结论 纤维蛋白胶联合血管外膜TIMP-1转染能够实现移植静脉TIMP-1基因的过表达;纤维蛋白胶外支架联合TIMP-1对移植静脉内膜和中膜增生有协同抑制作用.     

血管束植入治疗儿童股骨头坏死的远期疗效分析

目的:评价血管束植入治疗儿童股骨头坏死(Legg Calve Perthesdisease,LCPD)的远期疗效。方法:LCPD患儿27例,男22例,女5例;年龄8～12岁,平均10.5岁。治疗分为两组,Ⅰ组12例,男10例,女2例;年龄8～12岁,平均10.4岁;行多条血管束植入术合改良Chiari骨盆截骨延长术治疗。Ⅱ组15例,男12例,女3例;年龄9～12岁,平均10.6岁;单行改良Chiari骨盆截骨延长术治疗。均于术后3个月后戴外展支架下床承重行走,同时行患髋被动旋转推压疗法治疗。结果:随访5～8年,平均6年3个月。治疗后两组的髋臼-股骨头指数(AHI)均恢复正常,Ⅰ组出现的并发症较多。疗效评定采用Stulberg标准:Ⅰ组12例中优4例,良2例,中2例,可2例,差2例;Ⅱ组15例中优13例,良1例,中1例。经Fisher检验,有显著性差异(P<0.05),Ⅱ组治疗效果优于Ⅰ组。Ⅰ组的平均恢复时间(25.0±1.6)个月,Ⅱ组(14.0±0.9)个月,经t检验,有显著性差异(P<0.001)。结论:LCPD是一种自限性疾病,利用血管束植入术不能缩短LCPD的恢复时间,并且并发症多。对于包容不好的患者,手术包容治疗是治疗的关键。     

新型纤维素导管桥接周围神经缺损的研究

目的运用一种新型纤维素材料导管桥接周围神经缺损。方法将原代培养、纯化的Schwann细胞种植入新型复合纤维素导管作为移植物，于大鼠模型上桥接1．0cm坐骨神经缺损。术后1周，扫描电镜观察Schwann细胞在导管内壁的生长情况；术后12周，取桥接物中段,行半薄切片光镜观察及超薄切片电镜观察神经纤维再生状况。结果桥接物植入后1周,Schwann细胞贴附于导管内壁良好生长。植入后12周，管内有大量排列整齐、结构成熟的再生神经纤维。结论新型纤维素组织工程化神经可有效桥接周围神经缺损，该导管材料在医疗中具有广阔的应用前景。