保留软骨的去细胞全喉支架的实验研究
摘要
进展期喉癌或者下咽癌的患者常需实施全喉切除术，导致患者言语功能和吞咽功能受损。全喉切术后患者由于无法与人正常交流，常产生自卑，抑郁，焦虑的情绪，45%的全喉切除术后患者具有抑郁症状，远高于其他癌症患者。随着头颈外科手术技术的提高，放化疗的应用，喉癌的5年生存率逐年提高，喉癌的治疗重点应从单纯的追求提高患者生存率向提高患者的生存质量转变，要同时注重恢复和重建患者的全喉功能，提高患者的社会参与性和生活质量。全喉功能的重建成为喉癌术后患者和严重喉损伤患者最热切的临床治疗需要。
喉是由软骨，肌肉，黏膜，腺体组成的复合器官，目前传统的手术治疗方法无法完成全喉组织的重建。喉移植是目前对于全喉切除术后患者和严重喉损伤患者唯一有效的重建方法。全世界第一例全喉移植手术于1998年成功实施，移植喉可以为患者提供近似正常的发音，吞咽和呼吸功能。据报道有75%的喉癌切除术后患者愿意接受喉移植手术。然而第一例喉移植患者系严重喉损伤患者，为了避免免疫排斥，在移植术后的12年中长期服用免疫抑制剂。免疫抑制剂的应用常会导致感染，肿瘤复发或转移，大大限制了喉移植在喉癌术后患者身上的使用。
由于喉解剖结构的特殊性，目前没有任何一种天然的或者人工合成的材料能够模拟人喉的结构。组织工程学的研究为器官重建提供了新的思路，去细胞同种异体或者异种心脏，肺脏，肝脏支架的构建为组织工程器官提供新的支架结构。去细胞方法可构建出具有天然三维结构的支架，该支架复合种子细胞可构建出具有一定功能的组织或者器官。浸泡法构建的去细胞人喉支架已有报道，该种方法构建的组织工程气管已有移植报道，但是运用该种去细胞方法构建的支架会提高支架的顺应性，导致喉或气管支架软化，甚至导致移植术后喉气管狭窄。
喉的支撑结构主要在于喉软骨基质成份。喉软骨细胞具有合成和分泌软骨基质和胶原纤维的能力，因此喉软骨细胞的活性对于维持喉支架的三维框架结构至关重要。软骨是没有血管，没有神经的组织。软骨细胞主要通过基质的渗透作用吸收营养物质。软骨细胞被软骨基质严密包裹这样就避免了软骨细胞与外界的抗原物质相接触。因此通过血管灌注能够去除喉组织中高免疫原性的组织而较好的保留软骨细胞的活性。
直接的同种异体移植如果不使用免疫抑制剂会导致严重的免疫排斥反应。主要组织相容性抗原（MHC）的表达是导致免疫排斥反应的主要原因。在对人喉主要组织相容性抗原的研究发现人喉主要组织相容性抗原分布于黏膜上皮层，黏膜下腺和软骨膜，软骨基质中不表达主要组织相容性抗原，软骨组织在同种异体移植中不会引起免疫排斥反应。
本研究通过靶控麻醉泵向离体喉的颈外动脉中持续灌注去离子剂构建保留喉软骨活性的全喉支架。支架大体观察，组织学观察，显微结构观察，DNA含量分析，软骨细胞活性检测，支架免疫原性检测研究通过灌注法制备出结构完整，无免疫原性的全喉软骨三维支架的可行性；将去细胞全喉软骨支架埋植到受体大网膜内检测受体对支架的大体反应情况和淋巴细胞浸润情况，研究去细胞全喉软骨支架的生物相容性和免疫原性。将骨髓间充质干细胞体外培养扩增后种植到去细胞全喉软骨支架上，观察细胞在支架上的生长情况，证明支架的毒性和细胞相容性，细胞标记物示踪骨髓间充质干细胞在支架上的生长情况，为构建组织工程全喉提供理论依据。
第一部分保留软骨的去细胞全喉支架的体外研究
目的：通过靶控麻醉泵灌注去细胞液构建保留软骨的去细胞全喉支架，对支架进行体外研究，探索制备组织工程喉支架的可行性。
方法：实验动物雄性SD大鼠，体重200g左右，随机分为实验组和对照组，实验组分离颈总动脉，结扎分支，经颈总动脉通过靶控麻醉泵向甲状腺上动脉和甲状腺下动脉内持续灌注肝素生理盐水，1%SDS，1%Triton X-100，PBS等溶液；对照组，将喉组织从体内取出喉，不做任何处理，置于含青链霉素的PBS液中与实验组灌注等长的时间。所得标本行大体观察，组织学观察，扫描电镜观察，DNA含量分子检测，软骨活力检测，免疫学检测。
结果：全喉的去细胞灌注实验证明通过14h去细胞灌注处理后，全喉大体结构完整，喉组织颜色苍白透明，体积无明显变化，硬度无明显改变，喉的三维结构无明显改变。全喉支架去细胞后除软骨组织中的细胞外，其他组织如肌肉，黏膜，腺体等软组织中的细胞成份基本上被去除，去细胞彻底，效果明显。细胞外的基质成份被完整保留，组织结构清楚，层次分明。组织中的细胞成份基本上被去除，但全喉细胞外基质成份之间的网状连接依然存在，胶原束和细胞外基质成份组成的不规则腔隙存在于喉的会厌，声带，环状软骨，甲状软骨等表面。PCR实验结果显示，不含软骨成份的去细胞支架中几乎无GAPDH基因的扩增，而含软骨成份的去细胞软骨支架GAPDH基因的扩增远低于正常喉组织GAPDH基因的扩增。去细胞后甲状软骨，环状软骨中活的细胞并未见明显变化，组织绝大多数细胞处于成活状态，与对照组无显著差异。去细胞全喉基质成份MHC-Ⅰ和MHC-Ⅱ染色阴性。
结论：通过靶控麻醉泵持续给喉的双侧颈总动脉泵入去污剂，可以在较好的保留软骨细胞活性的同时去除喉肌肉，黏膜，腺体等组织中的细胞，构建出完整的全喉软骨框架结构，该支架的具有良好三维框架结构，较低的免疫原性和较好的机械力学结构，可作为全喉缺损修复的理想支架材料。
第二部分保留软骨的去细胞全喉支架的体内研究
目的：将去细胞全喉软骨支架植入同种异体受体动物腹腔内，观察受体对支架的反应，探讨支架的免疫原性和生物相容性。
方法：实验组将供体BN大鼠的离体喉按照实验一的制备方法经颈总动脉灌注后构建成去细胞全喉软骨支架，将支架埋植到受体SD大鼠的大网膜内；对照组将供体BN大鼠的离体喉不做任何处理直接埋植到受体SD大鼠的大网膜内；实验组和对照组于埋植后2周，4周，8周，12周后取出埋植的标本行大体观察，HE染色观察，CD3+T细胞和CD8+T细胞免疫组化观察。
结果：对照组和实验组各组动物均存活至各观察时间点，动物一般状况良好，伤口愈合良好，无明显的伤口裂开，渗血，溢脓等情况。所有的对照组均引起了强烈的免疫排斥反应，对照组喉被大网膜形成的纤维组织严密包裹，持续的炎性刺激使得纤维膜变得很厚。对照组可见大量的淋巴细胞增生，黏膜，腺体，肌肉，软骨组织受到了不同程度度破坏。CD8+T细胞和CD3+T细胞在各观察点的细胞数量在正常喉埋植后明显高于去细胞全喉软骨支架埋植体内后。去细胞喉支架埋植体内后并未引起强烈的免疫排斥反应。至各观察点取出包埋的喉支架发现，去细胞喉支架仅被薄层的大网膜包裹，未见明显的急性或慢性炎性反应。去细胞喉支架埋植体内4周后可见新生血管形成和成纤维样细胞增生。
结论：灌注法构建的去细胞全喉软骨支架埋植于同种异体大鼠的腹腔中，未引起强烈的免疫排斥反应，同时又有新生血管和成纤维样细胞的生成说明去细胞全喉软骨支架具有较好的生物相容性，能促进组织和器官的再生。
第三部分去细胞全喉软骨支架的再细胞化研究
目的：研究体外培养扩增骨髓间充质干细胞的方法，将骨髓间充质干细胞种植到去细胞支架表面，观察细胞在支架表面的生长情况，探讨去细胞全喉软骨支架再细胞化的可行性。
方法：采用密度梯度离心法结合差速贴壁法获取大鼠骨髓间充质干细胞，经免疫细胞化学法鉴定后，用去细胞全喉软骨支架的浸提液培养骨髓间充质干细胞，观察细胞的生长情况，了解支架的毒性。用CM-Dil标记的骨髓干细胞后以1×106的密度将骨髓间充质干细胞种植到支架的环甲肌，环勺后肌，声带等脱细胞基质中，完全培养基，37℃，5%二氧化碳中培养3天，5天，7天后生物显微镜，荧光显微镜，扫描电镜下观察细胞在支架上的生长情况。
结果：培养3代的骨髓间充质干细胞，细胞增殖速度快，传代的细胞形态较均一，大多数呈梭形，漩涡生长，保持良好的生长状态。免疫细胞化学结果显示CD29，CD44表达阳性，且阳性率大于90%，此结果符合骨髓间充质干细胞的鉴定标准。细胞可在支架的浸提液中正常生长。经过DM-DIl标记后，骨髓间充质干细胞的阳性标记率几乎可达到100%，将标记后的骨髓间充质干细胞种植到支架上，可见细胞分散在支架表面，但细胞分布成团状或者点状。去细胞全喉软骨支架种植骨髓干细胞后，组织学可见细胞主要生长于喉支架的内侧和外侧表面，细胞生长状态良好，分布均匀。扫描电镜结果显示，骨髓间充质干细胞种植到支架上3天可见少量细胞在支架上生长，相互之间伸出胞突，5天后可见细胞成片状分布在支架表面，7天后，支架表面形成了一层膜状结构，铺满整个支架。
结论：通过密度梯度离心法结合差速贴壁法培养，可获得纯化的骨髓间充质干细胞。将骨髓间充质干细胞种植到去细胞全喉软骨支架上可见细胞在支架上正常生长和增殖，提示支架是无毒的不会导致细胞死亡或者突变，支架材料与细胞支架具有良好的亲和性，去细胞支架具有较好的生物相容性。骨髓间充质干细胞可作为组织工程喉的种子细胞。
Patients with advanced laryngeal or hypopharyngeal cancer sometimes need totallaryngectomy which impaired their speech and swallowing function. Patients with totallaryngectomy cann’t talk with other people often feel self-esteem, depression and anxiety.It was report that about45%patients with total laryngectomy have depressive symptoms,which are much higher than the patients with other cancer. With improvement of the headand neck surgery technology, the5-year survival rate has increased. Laryngeal cancertreatment should focus on to improve the patient’s quality of life from the simple improvethe survival rate. We shoule focue on recover and reconstruct in patient’s laryngealfunction.Reconstruction of the laryngeal function became the most fervent clinicaltreatment need for patients with advanced laryngeal carcinoma and severe throat injury.
The larynx is a complex organ composed of cartilage, muscle, mucous membranes, and glands. Currently, there is no pratical way to reconstruct the comples structure of thelarynx by conventional surgery. Laryngeal transplantation is the only effectivereconstruction method for total laryngectomy and patients with severe laryngeal injury.The transplanted larynx can make the patient a near-normal voice and adequate swallowand respiration function. It has been reported that nearly75%laryngectomy patient willaccept a larynx transplant if offered. However, the first case of laryngeal transplantation ispatient with severe laryngeal injury, in order to avoid immune rejection aftertransplantation, long-term use of immunosuppressive agents in the following12years postoperation. Immunosuppressive agents which often lead to infection, tumor recurrence ormetastasis, greatly limited the wide spread use for patient with laryngeal cancer. Due tothe special nature of laryngeal anatomical structure, there is no natural or syntheticsubstitute that can mimic the anatomical structure of the larynx, recent advances in tissueengineering may offer a promising alternative to organ replacement. Decellularization ofallogeneic or xenogeneic donor organs such as the heart, liver, and lung provide anacellular, natural, three-dimensional (3D) biological scaffold material that can be seededwith selected cell populations. Preliminary studies in animal models have shown that a3D,decellularized matrix scaffold with seed cells can result in the formation of functionaltissue. A human decellularized laryngeal scaffold has also been made. However, studies onthe compliance of a decellularized trachea have shown that decellularization techniques,which have be used in decellularized tracheas and larynxes, can increase the complianceof the trachea, which will likely be at a high risk for tracheomalacia and potentially evenpostoperative stenosis when implanted in a recipient.
Maintenance of the laryngeal framework depends mainly on the cartilage matrix. Aschondrocytes play important roles in synthesizing and secreting cartilage matrices andcollagen fibers, viable chondrocytes are vital for maintaining the3D structure of thelaryngeal framework. Cartilage is an avascular structure, in which chondrocytes receivenutrition by diffusion through the matrix. The absence of direct vascularization andpresence of a dense proteoglycan-collagen matrix will insulate chondrocytes from hostantigens. Therefore, perfusion of decellularizing agents through the laryngeal vasculature might remove the higher immunogenic tissue of the larynx and preserve chondrocyticviability.
It is widely known that recipients of allografts tend to reject grafts without the use ofimmunosuppressive agents post-implantation. Immunogenicity depends largely on theexpression of major histocompatibility complex (MHC) antigens that can initiate organtransplantation rejection. Studies on the distribution of MHC-II antigens in the humanlarynx have shown that they were primarily found in mucosal surface epithelium,submucosal glands, and perichondrium, but not in thyroid chondrocytes or cartilagematrix, suggesting that the cartilage are not the major antigenic structures of the larynxand thus responsible for transplant rejection
We constructed a low-immunogenic, whole-laryngeal scaffold in with the viabilityof laryngeal cartilage of the scaffold were preserved, while the cellular elements of othertissue were decellularized using perfusion technology by pump. The general observation,histological observation, microstructure observation, DNA content analysis, chondrocyteactivity analysis shown that structural integrity, non-immunogenic laryngeal cartilagescaffolds can be constructed; the acellular laryngeal cartilage implants to receptoromentum is detected the biocompatibility and immunogenicity of decellularzied laryngealcartilage scaffold. The bone marrow mesenchymal stem cells in vitro were amplificatedand seeded on the decellularized laryngeal cartilage scaffold. The cells could growth onthe scaffold. It could prove the toxicity and cell compatibility of the scaffold. Cell markedbone marrow mesenchymal stem cells could grow well on the scaffold, which couldprovide a theoretical basis for tissue engineering.
Part one: In vitro study of the decellularized cartilage laryngeal scaffold
[Objective] Constructe decellularized cartilage laryngeal scaffold by perfusion thedetergent through the Target-controlled infusion pump and in vitro studies scaffold, toexplore the feasibility of the preparation of tissue engineering laryngeal scaffold.
[Methods] Male, SD rats, weighing200g, were randomly divided into experimental group and control group.The experimental group, separate and ligate the carotid arterybranches, then perfuse the detergent via the carotid artery to the superior thyroid arteryand the inferior thyroid artery by target-controlled infusion pump. The perfusion sequenseis heparin physiological solution,1%SDS,1%Triton X-100, PBS; The control group, thelaryngeal tissue removed from the body throat, without any treatment, just dipping in aPBS solution containing streptomycin and penicillin.The persistence time is as well as theperfusion time. Both the experment Specimens and control group specimens wereobserved by gross observation, histological observation, scanning electron microscopy,DNA content molecules, cartilage viability testing, and immunological detection.
[Results] The images of the decellularized larynxes confirmed the efficacy of thedecellularization process in removing the majority of the cellular element. After14h ofperfusion with SDS, the larynxes appeared to be translucent and dilated, and retained theirshapes and stiff consistency. The treatment for decellularizing removed most of cellularcomponents from the mucosal and laryngeal muscle layers, and relatively preserved theextracellular matrix and laryngeal cartilage. The3D architecture of the larynx was notaltered. SEM demonstrated that all cellular components were removed, but the wholeorgan ECM network was preserved. After decellularzation, collagen bundles and ECMwith irregular luminal can be seen on the surface of laryngeal scaffolds (Epiglottis, falsevocal cord, vocal cord, cricoids and thyroid cartilage DNA analysis showed that thehousekeeping gene in the decellularized, cartilage-free, laryngeal sample were notamplified, but the GAPDH was amplified in the decellularized laryngeal samples withpreserved cartilage. The average DNA content of the decellularized laryngeal sampleswith preserved cartilages was less than that in the fresh laryngeal samples. Thechondrocyte viability of thyroid cartilage and cricoid cartilage were not changed greatly.Viable chondrocytes were found in all decellularized laryngeal cartilage layers. There wasno significant difference in the survival rate between the decellularized laryngeal cartilageand the fresh laryngeal cartilage.The decellularized larynx did not show the presence ofthe markers of MHC-I and MHC-II on the decellularized matrix.
[Coclusions] The perfusion of decellularization detergents can construct adecellularized, whole-laryngeal scaffold while preserving the cartilage. The other cellularcomponents in soft tissue were mostly removed during this process. The decellularizedwhole-laryngeal scaffold had an excellent anatomical structure and might be a good sourceof tissue engineering for scaffolds or frameworks for laryngeal reconstruction and offers apromising reconstruction material for patients.
Part two: In vivo study of the decellularized cartilage laryngeal scaffold
[Objectives] Implanted the decellularized laryngeal scaffold into the allograftrecipient animals’ abdominal cavity and observed the response of the receptors on thedecellularized laryngeal scaffold. Explore the immunogenicity and biocompatibility of thescaffold.
[Methods] The experiment group: put the BN rat perfution decellularized laryngealscaffold which was made as the part one described, on the receptor SD rats’ greateromentum. The control group: put the BN rat’s fresh larynx on the the receptor SD rats’greater omentum without any treatment. The experimental and control groups after2weeks,4weeks,8weeks,12weeks implantation, explanted the specimens for grossobservation, HE staining, CD3+T cells and CD8+T cell immunohistochemistry.
[Results] Both the control group animals and experimental group animals allsurvived to each observation time, the animals are generally in good condition, goodwound healing, and no obvious wound dehiscence, bleeding, pus overflow situation. All ofthe implanted fresh larynx samples showed strong immunological rejection. The freshlarynxes were surrounded by fibrous capsules and greater omentum. The greater omentumwhich encapsulated the fresh larynxes became thicker weeks after implantation. Thecontrol group had shown that the lymphocytic infiltration in the external and internal ofthe implanted larynxes. Mucosa, glands, muscles, cartilage tissue were damaged bydifferent degree. Compared to the implanted fresh larynxes, the implanted, decellularizedlarynxes were covered by a thinner greater omentum and there were no signs of acute orchronic rejection. Neovascularization and fibroblaset-like cells were observed on the decellularized laryngeal scaffold4weeks post-implantation.
[Conclusions] The perfusion decellularized laryngeal scaffold implanted in thereceport’s peritoneal cavity did not elicit strong immune rejection.At the same time therewere neovascularization and fibroblaset-like cells on the decellularized laryngeal scaffoldpost-implantation. This indicated that the decellulatized laryngeal scaffold with good cellbiocompatibility could promote the tissue and organ regeneration.Part three: Recellularization of the decellularized cartilage laryngeal scaffold
[Objective] To investigate the methods of isolation, culture and identification of ratbone marrow mesenchymal stem cells (BMMSCs). Seeded the BMMSCs on thedecellularized cartilage laryngeal scaffold, observe the growth of the BMMSCs. Explorethe feasibility of the decellularized cartilage laryngeal scaffolds’recellularization.
[Methods] BMMSCs were isolated from bone marrow of SD rats by desity gradientcentrifugation and differential ashesion methods. Cells were identificated by immunecytochemistry methods. The leach liquor of the decellularized laryngeal scaffold was usedto culture cells to observe the state of the cells and understand the toxicity of the scaffold.The BMMSc labeled by CM-Dil were seed on decellularized matrix of cricothyroid,posterior crico-arytenoid muscle and voval cord by the density1×106.The scaffold wasculture in37℃，5%CO2incubator for3days,5days7days. The histological, fluorescencemicroscopy, scanning electron microscopy were used to observe cell growth on thescaffold.
[Results] The third generations BMMCs were growing fast. The morphology of thepassaged cell is relatively uniform.Most of them are spindle-shape, whirlpool growth,maintain a good growth state.Immuncytochemistry results showed that CD29and CD44positive. The positive rate is more than90%. The results is in coincidence with thestandards of BMMSc.The BMMSc have a good growth rate in the leaching liguor. AfterDM-Dil markers, bone marrow mesenchymal stem cells positive mark rate almost canreach100%.The CM-Dil-labeled BMMSc seeded on decellurized scaffold dispersed onthe surface of the stent.The seeded cells on the decellulatied scaffold can be observed growth on the outside and inside of the scaffold by histology study.The cells were in goodstate and evenly distributed.The scanning electron microscope showed that BMMSc wereshown that a small amount of cells were grown and extended between cells after3dayspost seeded.After5days seeded, the cells were distributed like a sheet on the surface ofthe scaffold. After7days, the cells on the surface of the scaffold is formed a layer offilm-like structure on the scaffold.
[Conclusions] The pured BMMSc can be acquired by desity gradient centrifugationand differential ashesion method. The BMMSc seeded on the decellularized scaffold couldhave a good growth rate and generation. This could demonstrate that the decellularizedscaffold is non-toxic and does not cause cell death or mutation. There is a good affinitybetween the cells and the scaffold. The decellularized scaffold has a good biocompatibility.BMMSc can be used as seed cells for tissue engineering larynx.
喉是由软骨，肌肉，黏膜，腺体组成的复合器官，目前传统的手术治疗方法无法完成全喉组织的重建。喉移植是目前对于全喉切除术后患者和严重喉损伤患者唯一有效的重建方法。全世界第一例全喉移植手术于1998年成功实施，移植喉可以为患者提供近似正常的发音，吞咽和呼吸功能。据报道有75%的喉癌切除术后患者愿意接受喉移植手术。然而第一例喉移植患者系严重喉损伤患者，为了避免免疫排斥，在移植术后的12年中长期服用免疫抑制剂。免疫抑制剂的应用常会导致感染，肿瘤复发或转移，大大限制了喉移植在喉癌术后患者身上的使用。
由于喉解剖结构的特殊性，目前没有任何一种天然的或者人工合成的材料能够模拟人喉的结构。组织工程学的研究为器官重建提供了新的思路，去细胞同种异体或者异种心脏，肺脏，肝脏支架的构建为组织工程器官提供新的支架结构。去细胞方法可构建出具有天然三维结构的支架，该支架复合种子细胞可构建出具有一定功能的组织或者器官。浸泡法构建的去细胞人喉支架已有报道，该种方法构建的组织工程气管已有移植报道，但是运用该种去细胞方法构建的支架会提高支架的顺应性，导致喉或气管支架软化，甚至导致移植术后喉气管狭窄。
喉的支撑结构主要在于喉软骨基质成份。喉软骨细胞具有合成和分泌软骨基质和胶原纤维的能力，因此喉软骨细胞的活性对于维持喉支架的三维框架结构至关重要。软骨是没有血管，没有神经的组织。软骨细胞主要通过基质的渗透作用吸收营养物质。软骨细胞被软骨基质严密包裹这样就避免了软骨细胞与外界的抗原物质相接触。因此通过血管灌注能够去除喉组织中高免疫原性的组织而较好的保留软骨细胞的活性。
直接的同种异体移植如果不使用免疫抑制剂会导致严重的免疫排斥反应。主要组织相容性抗原（MHC）的表达是导致免疫排斥反应的主要原因。在对人喉主要组织相容性抗原的研究发现人喉主要组织相容性抗原分布于黏膜上皮层，黏膜下腺和软骨膜，软骨基质中不表达主要组织相容性抗原，软骨组织在同种异体移植中不会引起免疫排斥反应。
本研究通过靶控麻醉泵向离体喉的颈外动脉中持续灌注去离子剂构建保留喉软骨活性的全喉支架。支架大体观察，组织学观察，显微结构观察，DNA含量分析，软骨细胞活性检测，支架免疫原性检测研究通过灌注法制备出结构完整，无免疫原性的全喉软骨三维支架的可行性；将去细胞全喉软骨支架埋植到受体大网膜内检测受体对支架的大体反应情况和淋巴细胞浸润情况，研究去细胞全喉软骨支架的生物相容性和免疫原性。将骨髓间充质干细胞体外培养扩增后种植到去细胞全喉软骨支架上，观察细胞在支架上的生长情况，证明支架的毒性和细胞相容性，细胞标记物示踪骨髓间充质干细胞在支架上的生长情况，为构建组织工程全喉提供理论依据。
第一部分保留软骨的去细胞全喉支架的体外研究
目的：通过靶控麻醉泵灌注去细胞液构建保留软骨的去细胞全喉支架，对支架进行体外研究，探索制备组织工程喉支架的可行性。
方法：实验动物雄性SD大鼠，体重200g左右，随机分为实验组和对照组，实验组分离颈总动脉，结扎分支，经颈总动脉通过靶控麻醉泵向甲状腺上动脉和甲状腺下动脉内持续灌注肝素生理盐水，1%SDS，1%Triton X-100，PBS等溶液；对照组，将喉组织从体内取出喉，不做任何处理，置于含青链霉素的PBS液中与实验组灌注等长的时间。所得标本行大体观察，组织学观察，扫描电镜观察，DNA含量分子检测，软骨活力检测，免疫学检测。
结果：全喉的去细胞灌注实验证明通过14h去细胞灌注处理后，全喉大体结构完整，喉组织颜色苍白透明，体积无明显变化，硬度无明显改变，喉的三维结构无明显改变。全喉支架去细胞后除软骨组织中的细胞外，其他组织如肌肉，黏膜，腺体等软组织中的细胞成份基本上被去除，去细胞彻底，效果明显。细胞外的基质成份被完整保留，组织结构清楚，层次分明。组织中的细胞成份基本上被去除，但全喉细胞外基质成份之间的网状连接依然存在，胶原束和细胞外基质成份组成的不规则腔隙存在于喉的会厌，声带，环状软骨，甲状软骨等表面。PCR实验结果显示，不含软骨成份的去细胞支架中几乎无GAPDH基因的扩增，而含软骨成份的去细胞软骨支架GAPDH基因的扩增远低于正常喉组织GAPDH基因的扩增。去细胞后甲状软骨，环状软骨中活的细胞并未见明显变化，组织绝大多数细胞处于成活状态，与对照组无显著差异。去细胞全喉基质成份MHC-Ⅰ和MHC-Ⅱ染色阴性。
结论：通过靶控麻醉泵持续给喉的双侧颈总动脉泵入去污剂，可以在较好的保留软骨细胞活性的同时去除喉肌肉，黏膜，腺体等组织中的细胞，构建出完整的全喉软骨框架结构，该支架的具有良好三维框架结构，较低的免疫原性和较好的机械力学结构，可作为全喉缺损修复的理想支架材料。
第二部分保留软骨的去细胞全喉支架的体内研究
目的：将去细胞全喉软骨支架植入同种异体受体动物腹腔内，观察受体对支架的反应，探讨支架的免疫原性和生物相容性。
方法：实验组将供体BN大鼠的离体喉按照实验一的制备方法经颈总动脉灌注后构建成去细胞全喉软骨支架，将支架埋植到受体SD大鼠的大网膜内；对照组将供体BN大鼠的离体喉不做任何处理直接埋植到受体SD大鼠的大网膜内；实验组和对照组于埋植后2周，4周，8周，12周后取出埋植的标本行大体观察，HE染色观察，CD3+T细胞和CD8+T细胞免疫组化观察。
结果：对照组和实验组各组动物均存活至各观察时间点，动物一般状况良好，伤口愈合良好，无明显的伤口裂开，渗血，溢脓等情况。所有的对照组均引起了强烈的免疫排斥反应，对照组喉被大网膜形成的纤维组织严密包裹，持续的炎性刺激使得纤维膜变得很厚。对照组可见大量的淋巴细胞增生，黏膜，腺体，肌肉，软骨组织受到了不同程度度破坏。CD8+T细胞和CD3+T细胞在各观察点的细胞数量在正常喉埋植后明显高于去细胞全喉软骨支架埋植体内后。去细胞喉支架埋植体内后并未引起强烈的免疫排斥反应。至各观察点取出包埋的喉支架发现，去细胞喉支架仅被薄层的大网膜包裹，未见明显的急性或慢性炎性反应。去细胞喉支架埋植体内4周后可见新生血管形成和成纤维样细胞增生。
结论：灌注法构建的去细胞全喉软骨支架埋植于同种异体大鼠的腹腔中，未引起强烈的免疫排斥反应，同时又有新生血管和成纤维样细胞的生成说明去细胞全喉软骨支架具有较好的生物相容性，能促进组织和器官的再生。
第三部分去细胞全喉软骨支架的再细胞化研究
目的：研究体外培养扩增骨髓间充质干细胞的方法，将骨髓间充质干细胞种植到去细胞支架表面，观察细胞在支架表面的生长情况，探讨去细胞全喉软骨支架再细胞化的可行性。
方法：采用密度梯度离心法结合差速贴壁法获取大鼠骨髓间充质干细胞，经免疫细胞化学法鉴定后，用去细胞全喉软骨支架的浸提液培养骨髓间充质干细胞，观察细胞的生长情况，了解支架的毒性。用CM-Dil标记的骨髓干细胞后以1×106的密度将骨髓间充质干细胞种植到支架的环甲肌，环勺后肌，声带等脱细胞基质中，完全培养基，37℃，5%二氧化碳中培养3天，5天，7天后生物显微镜，荧光显微镜，扫描电镜下观察细胞在支架上的生长情况。
结果：培养3代的骨髓间充质干细胞，细胞增殖速度快，传代的细胞形态较均一，大多数呈梭形，漩涡生长，保持良好的生长状态。免疫细胞化学结果显示CD29，CD44表达阳性，且阳性率大于90%，此结果符合骨髓间充质干细胞的鉴定标准。细胞可在支架的浸提液中正常生长。经过DM-DIl标记后，骨髓间充质干细胞的阳性标记率几乎可达到100%，将标记后的骨髓间充质干细胞种植到支架上，可见细胞分散在支架表面，但细胞分布成团状或者点状。去细胞全喉软骨支架种植骨髓干细胞后，组织学可见细胞主要生长于喉支架的内侧和外侧表面，细胞生长状态良好，分布均匀。扫描电镜结果显示，骨髓间充质干细胞种植到支架上3天可见少量细胞在支架上生长，相互之间伸出胞突，5天后可见细胞成片状分布在支架表面，7天后，支架表面形成了一层膜状结构，铺满整个支架。
结论：通过密度梯度离心法结合差速贴壁法培养，可获得纯化的骨髓间充质干细胞。将骨髓间充质干细胞种植到去细胞全喉软骨支架上可见细胞在支架上正常生长和增殖，提示支架是无毒的不会导致细胞死亡或者突变，支架材料与细胞支架具有良好的亲和性，去细胞支架具有较好的生物相容性。骨髓间充质干细胞可作为组织工程喉的种子细胞。
Patients with advanced laryngeal or hypopharyngeal cancer sometimes need totallaryngectomy which impaired their speech and swallowing function. Patients with totallaryngectomy cann’t talk with other people often feel self-esteem, depression and anxiety.It was report that about45%patients with total laryngectomy have depressive symptoms,which are much higher than the patients with other cancer. With improvement of the headand neck surgery technology, the5-year survival rate has increased. Laryngeal cancertreatment should focus on to improve the patient’s quality of life from the simple improvethe survival rate. We shoule focue on recover and reconstruct in patient’s laryngealfunction.Reconstruction of the laryngeal function became the most fervent clinicaltreatment need for patients with advanced laryngeal carcinoma and severe throat injury.
The larynx is a complex organ composed of cartilage, muscle, mucous membranes, and glands. Currently, there is no pratical way to reconstruct the comples structure of thelarynx by conventional surgery. Laryngeal transplantation is the only effectivereconstruction method for total laryngectomy and patients with severe laryngeal injury.The transplanted larynx can make the patient a near-normal voice and adequate swallowand respiration function. It has been reported that nearly75%laryngectomy patient willaccept a larynx transplant if offered. However, the first case of laryngeal transplantation ispatient with severe laryngeal injury, in order to avoid immune rejection aftertransplantation, long-term use of immunosuppressive agents in the following12years postoperation. Immunosuppressive agents which often lead to infection, tumor recurrence ormetastasis, greatly limited the wide spread use for patient with laryngeal cancer. Due tothe special nature of laryngeal anatomical structure, there is no natural or syntheticsubstitute that can mimic the anatomical structure of the larynx, recent advances in tissueengineering may offer a promising alternative to organ replacement. Decellularization ofallogeneic or xenogeneic donor organs such as the heart, liver, and lung provide anacellular, natural, three-dimensional (3D) biological scaffold material that can be seededwith selected cell populations. Preliminary studies in animal models have shown that a3D,decellularized matrix scaffold with seed cells can result in the formation of functionaltissue. A human decellularized laryngeal scaffold has also been made. However, studies onthe compliance of a decellularized trachea have shown that decellularization techniques,which have be used in decellularized tracheas and larynxes, can increase the complianceof the trachea, which will likely be at a high risk for tracheomalacia and potentially evenpostoperative stenosis when implanted in a recipient.
Maintenance of the laryngeal framework depends mainly on the cartilage matrix. Aschondrocytes play important roles in synthesizing and secreting cartilage matrices andcollagen fibers, viable chondrocytes are vital for maintaining the3D structure of thelaryngeal framework. Cartilage is an avascular structure, in which chondrocytes receivenutrition by diffusion through the matrix. The absence of direct vascularization andpresence of a dense proteoglycan-collagen matrix will insulate chondrocytes from hostantigens. Therefore, perfusion of decellularizing agents through the laryngeal vasculature might remove the higher immunogenic tissue of the larynx and preserve chondrocyticviability.
It is widely known that recipients of allografts tend to reject grafts without the use ofimmunosuppressive agents post-implantation. Immunogenicity depends largely on theexpression of major histocompatibility complex (MHC) antigens that can initiate organtransplantation rejection. Studies on the distribution of MHC-II antigens in the humanlarynx have shown that they were primarily found in mucosal surface epithelium,submucosal glands, and perichondrium, but not in thyroid chondrocytes or cartilagematrix, suggesting that the cartilage are not the major antigenic structures of the larynxand thus responsible for transplant rejection
We constructed a low-immunogenic, whole-laryngeal scaffold in with the viabilityof laryngeal cartilage of the scaffold were preserved, while the cellular elements of othertissue were decellularized using perfusion technology by pump. The general observation,histological observation, microstructure observation, DNA content analysis, chondrocyteactivity analysis shown that structural integrity, non-immunogenic laryngeal cartilagescaffolds can be constructed; the acellular laryngeal cartilage implants to receptoromentum is detected the biocompatibility and immunogenicity of decellularzied laryngealcartilage scaffold. The bone marrow mesenchymal stem cells in vitro were amplificatedand seeded on the decellularized laryngeal cartilage scaffold. The cells could growth onthe scaffold. It could prove the toxicity and cell compatibility of the scaffold. Cell markedbone marrow mesenchymal stem cells could grow well on the scaffold, which couldprovide a theoretical basis for tissue engineering.
Part one: In vitro study of the decellularized cartilage laryngeal scaffold
[Objective] Constructe decellularized cartilage laryngeal scaffold by perfusion thedetergent through the Target-controlled infusion pump and in vitro studies scaffold, toexplore the feasibility of the preparation of tissue engineering laryngeal scaffold.
[Methods] Male, SD rats, weighing200g, were randomly divided into experimental group and control group.The experimental group, separate and ligate the carotid arterybranches, then perfuse the detergent via the carotid artery to the superior thyroid arteryand the inferior thyroid artery by target-controlled infusion pump. The perfusion sequenseis heparin physiological solution,1%SDS,1%Triton X-100, PBS; The control group, thelaryngeal tissue removed from the body throat, without any treatment, just dipping in aPBS solution containing streptomycin and penicillin.The persistence time is as well as theperfusion time. Both the experment Specimens and control group specimens wereobserved by gross observation, histological observation, scanning electron microscopy,DNA content molecules, cartilage viability testing, and immunological detection.
[Results] The images of the decellularized larynxes confirmed the efficacy of thedecellularization process in removing the majority of the cellular element. After14h ofperfusion with SDS, the larynxes appeared to be translucent and dilated, and retained theirshapes and stiff consistency. The treatment for decellularizing removed most of cellularcomponents from the mucosal and laryngeal muscle layers, and relatively preserved theextracellular matrix and laryngeal cartilage. The3D architecture of the larynx was notaltered. SEM demonstrated that all cellular components were removed, but the wholeorgan ECM network was preserved. After decellularzation, collagen bundles and ECMwith irregular luminal can be seen on the surface of laryngeal scaffolds (Epiglottis, falsevocal cord, vocal cord, cricoids and thyroid cartilage DNA analysis showed that thehousekeeping gene in the decellularized, cartilage-free, laryngeal sample were notamplified, but the GAPDH was amplified in the decellularized laryngeal samples withpreserved cartilage. The average DNA content of the decellularized laryngeal sampleswith preserved cartilages was less than that in the fresh laryngeal samples. Thechondrocyte viability of thyroid cartilage and cricoid cartilage were not changed greatly.Viable chondrocytes were found in all decellularized laryngeal cartilage layers. There wasno significant difference in the survival rate between the decellularized laryngeal cartilageand the fresh laryngeal cartilage.The decellularized larynx did not show the presence ofthe markers of MHC-I and MHC-II on the decellularized matrix.
[Coclusions] The perfusion of decellularization detergents can construct adecellularized, whole-laryngeal scaffold while preserving the cartilage. The other cellularcomponents in soft tissue were mostly removed during this process. The decellularizedwhole-laryngeal scaffold had an excellent anatomical structure and might be a good sourceof tissue engineering for scaffolds or frameworks for laryngeal reconstruction and offers apromising reconstruction material for patients.
Part two: In vivo study of the decellularized cartilage laryngeal scaffold
[Objectives] Implanted the decellularized laryngeal scaffold into the allograftrecipient animals’ abdominal cavity and observed the response of the receptors on thedecellularized laryngeal scaffold. Explore the immunogenicity and biocompatibility of thescaffold.
[Methods] The experiment group: put the BN rat perfution decellularized laryngealscaffold which was made as the part one described, on the receptor SD rats’ greateromentum. The control group: put the BN rat’s fresh larynx on the the receptor SD rats’greater omentum without any treatment. The experimental and control groups after2weeks,4weeks,8weeks,12weeks implantation, explanted the specimens for grossobservation, HE staining, CD3+T cells and CD8+T cell immunohistochemistry.
[Results] Both the control group animals and experimental group animals allsurvived to each observation time, the animals are generally in good condition, goodwound healing, and no obvious wound dehiscence, bleeding, pus overflow situation. All ofthe implanted fresh larynx samples showed strong immunological rejection. The freshlarynxes were surrounded by fibrous capsules and greater omentum. The greater omentumwhich encapsulated the fresh larynxes became thicker weeks after implantation. Thecontrol group had shown that the lymphocytic infiltration in the external and internal ofthe implanted larynxes. Mucosa, glands, muscles, cartilage tissue were damaged bydifferent degree. Compared to the implanted fresh larynxes, the implanted, decellularizedlarynxes were covered by a thinner greater omentum and there were no signs of acute orchronic rejection. Neovascularization and fibroblaset-like cells were observed on the decellularized laryngeal scaffold4weeks post-implantation.
[Conclusions] The perfusion decellularized laryngeal scaffold implanted in thereceport’s peritoneal cavity did not elicit strong immune rejection.At the same time therewere neovascularization and fibroblaset-like cells on the decellularized laryngeal scaffoldpost-implantation. This indicated that the decellulatized laryngeal scaffold with good cellbiocompatibility could promote the tissue and organ regeneration.Part three: Recellularization of the decellularized cartilage laryngeal scaffold
[Objective] To investigate the methods of isolation, culture and identification of ratbone marrow mesenchymal stem cells (BMMSCs). Seeded the BMMSCs on thedecellularized cartilage laryngeal scaffold, observe the growth of the BMMSCs. Explorethe feasibility of the decellularized cartilage laryngeal scaffolds’recellularization.
[Methods] BMMSCs were isolated from bone marrow of SD rats by desity gradientcentrifugation and differential ashesion methods. Cells were identificated by immunecytochemistry methods. The leach liquor of the decellularized laryngeal scaffold was usedto culture cells to observe the state of the cells and understand the toxicity of the scaffold.The BMMSc labeled by CM-Dil were seed on decellularized matrix of cricothyroid,posterior crico-arytenoid muscle and voval cord by the density1×106.The scaffold wasculture in37℃，5%CO2incubator for3days,5days7days. The histological, fluorescencemicroscopy, scanning electron microscopy were used to observe cell growth on thescaffold.
[Results] The third generations BMMCs were growing fast. The morphology of thepassaged cell is relatively uniform.Most of them are spindle-shape, whirlpool growth,maintain a good growth state.Immuncytochemistry results showed that CD29and CD44positive. The positive rate is more than90%. The results is in coincidence with thestandards of BMMSc.The BMMSc have a good growth rate in the leaching liguor. AfterDM-Dil markers, bone marrow mesenchymal stem cells positive mark rate almost canreach100%.The CM-Dil-labeled BMMSc seeded on decellurized scaffold dispersed onthe surface of the stent.The seeded cells on the decellulatied scaffold can be observed growth on the outside and inside of the scaffold by histology study.The cells were in goodstate and evenly distributed.The scanning electron microscope showed that BMMSc wereshown that a small amount of cells were grown and extended between cells after3dayspost seeded.After5days seeded, the cells were distributed like a sheet on the surface ofthe scaffold. After7days, the cells on the surface of the scaffold is formed a layer offilm-like structure on the scaffold.
[Conclusions] The pured BMMSc can be acquired by desity gradient centrifugationand differential ashesion method. The BMMSc seeded on the decellularized scaffold couldhave a good growth rate and generation. This could demonstrate that the decellularizedscaffold is non-toxic and does not cause cell death or mutation. There is a good affinitybetween the cells and the scaffold. The decellularized scaffold has a good biocompatibility.BMMSc can be used as seed cells for tissue engineering larynx.
引文
[1] Armstrong W B, Netterville J L. Anatomy of the larynx, trachea, and bronchi[J].Otolaryngol Clin North Am,1995,28(4):685～699.
[2] Minnich D J, Mathisen D J. Anatomy of the trachea, carina, and bronchi[J]. ThoracSurg Clin,2007,17(4):571～585.
[3] Macchiarini P, Jungebluth P, Go T, et al. Clinical transplantation of atissue-engineered airway[J]. Lancet,2008,372(9655):2023～2030.
[4] Yamashita M, Hirano S, Kanemaru S, et al. Side population cells in the human vocalfold[J]. Ann Otol Rhinol Laryngol,2007,116(11):847～852.
[5] Goodell M A, Brose K, Paradis G, et al. Isolation and functional properties ofmurine hematopoietic stem cells that are replicating in vivo[J]. J Exp Med,1996,183(4):1797～1806.
[6] Gugatschka M, Kojima T, Ohno S, et al. Recruitment patterns of side populationcells during wound healing in rat vocal folds[J]. Laryngoscope,2011,121(8):1662～1667.
[7] Hanson S E, Kim J, Johnson B H, et al. Characterization of mesenchymal stem cellsfrom human vocal fold fibroblasts[J]. Laryngoscope,2010,120(3):546～551.
[8] Gugatschka M, Ohno S, Saxena A, et al. Regenerative medicine of the larynx.Where are we today? A review[J]. J Voice,2012,26(5):670～677.
[9] Hirano S, Kishimoto Y, Suehiro A, et al. Regeneration of Aged Vocal Fold: FirstHuman Case Treated With Fibroblast Growth Factor[J]. The Laryngoscope,2008,118(12):2254～2259.
[10] Hirano S, Kishimoto Y, Suehiro A, et al. Regeneration of aged vocal fold: Firsthuman case treated with fibroblast growth factor[J]. The Laryngoscope,2009,119(1):197～202.
[11] Hirano S, Nagai H, Tateya I, et al. Regeneration of aged vocal folds with basicfibroblast growth factor in a rat model: a preliminary report[J]. Ann Otol RhinolLaryngol,2005,114(4):304～308.
[12] Ohno T, Hirano S, Rousseau B. Gene expression of transforming growthfactor-beta1and hepatocyte growth factor during wound healing of injured ratvocal fold[J]. Laryngoscope,2009,119(4):806～810.
[13] Ohno T, Yoo M J, Swanson E R, et al. Regenerative effects of basic fibroblastgrowth factor on extracellular matrix production in aged rat vocal folds[J]. Ann OtolRhinol Laryngol,2009,118(8):559～564.
[14] Matsumoto K, Nakamura T. Hepatocyte growth factor (HGF) as a tissue organizerfor organogenesis and regeneration[J]. Biochem Biophys Res Commun,1997,239(3):639～644.
[15] Hirano S, Bless D, Heisey D, et al. Roles of hepatocyte growth factor andtransforming growth factor beta1in production of extracellular matrix by caninevocal fold fibroblasts[J]. Laryngoscope,2003,113(1):144～148.
[16] Hirano S, Bless D M, Nagai H, et al. Growth factor therapy for vocal fold scarring ina canine model[J]. Ann Otol Rhinol Laryngol,2004,113(10):777～785.
[17] Ohno T, Hirano S, Kanemaru S, et al. Drug delivery system of hepatocyte growthfactor for the treatment of vocal fold scarring in a canine model[J]. Ann OtolRhinol Laryngol,2007,116(10):762～769.
[18] Sheen-Chen S M, Liu Y W, Eng H L, et al. Serum levels of hepatocyte growthfactor in patients with breast cancer[J]. Cancer Epidemiol Biomarkers Prev,2005,14(3):715～717.
[19] Farran A J, Teller S S, Jha A K, et al. Effects of matrix composition, microstructure,and viscoelasticity on the behaviors of vocal fold fibroblasts cultured inthree-dimensional hydrogel networks[J]. Tissue Eng Part A,2010,16(4):1247～1261.
[20] Butler J E, Hammond T H, Gray S D. Gender-related differences of hyaluronic aciddistribution in the human vocal fold[J]. Laryngoscope,2001,111(5):907～911.
[21] Munoz-Pinto D J, Jimenez-Vergara A C, Gelves L M, et al. Probing vocal foldfibroblast response to hyaluronan in3D contexts[J]. Biotechnol Bioeng,2009,104(4):821～831.
[22] Luo Y, Kobler J B, Zeitels S M, et al. Effects of growth factors on extracellularmatrix production by vocal fold fibroblasts in3-dimensional culture[J]. Tissue Eng,2006,12(12):3365～3374.
[23] Park H, Karajanagi S, Wolak K, et al. Three-dimensional hydrogel model usingadipose-derived stem cells for vocal fold augmentation[J]. Tissue Eng Part A,2010,16(2):535～543.
[24] Luo Y, Kobler J B, Heaton J T, et al. Injectable hyaluronic acid-dextran hydrogelsand effects of implantation in ferret vocal fold[J]. J Biomed Mater Res B ApplBiomater,2010,93(2):386～393.
[25] Webb K, Hitchcock R W, Smeal R M, et al. Cyclic strain increases fibroblastproliferation, matrix accumulation, and elastic modulus of fibroblast-seededpolyurethane constructs[J]. J Biomech,2006,39(6):1136～1144.
[26] Webb K, Li W, Hitchcock R W, et al. Comparison of human fibroblast ECM-relatedgene expression on elastic three-dimensional substrates relative to two-dimensionalfilms of the same material[J]. Biomaterials,2003,24(25):4681～4690.
[27] Thibeault S L, Duflo S. Inflammatory cytokine responses to synthetic extracellularmatrix injection to the vocal fold lamina propria[J]. Ann Otol Rhinol Laryngol,2008,117(3):221～226.
[28] Hahn M S, Teply B A, Stevens M M, et al. Collagen composite hydrogels for vocalfold lamina propria restoration[J]. Biomaterials,2006,27(7):1104～1109.
[29] Huber J E, Spievack A, Simmons-Byrd A, et al. Extracellular matrix as a scaffoldfor laryngeal reconstruction[J]. Ann Otol Rhinol Laryngol,2003,112(5):428～433.
[30] Xu C C, Chan R W. Pore architecture of a bovine acellular vocal fold scaffold[J].Tissue Eng Part A,2008,14(11):1893～1903.
[31] Xu C C, Chan R W, Weinberger D G, et al. A bovine acellular scaffold for vocalfold reconstruction in a rat model[J]. J Biomed Mater Res A,2010,92(1):18～32.
[32] Chan R W, Rodriguez M L, McFetridge P S. The human umbilical vein withWharton's jelly as an allogeneic, acellular construct for vocal fold restoration[J].Tissue Eng Part A,2009,15(11):3537～3546.
[33] Wei H J, Liang H C, Lee M H, et al. Construction of varying porous structures inacellular bovine pericardia as a tissue-engineering extracellular matrix[J].Biomaterials,2005,26(14):1905～1913.
[34] Xu C C, Chan R W, Weinberger D G, et al. Controlled release of hepatocyte growthfactor from a bovine acellular scaffold for vocal fold reconstruction[J]. J BiomedMater Res A,2010,93(4):1335～1347.
[35] Chhetri D K, Head C, Revazova E, et al. Lamina propria replacement therapy withcultured autologous fibroblasts for vocal fold scars[J]. Otolaryngol Head Neck Surg,2004,131(6):864～870.
[36] Kanemaru S, Nakamura T, Omori K, et al. Regeneration of the vocal fold usingautologous mesenchymal stem cells[J]. Ann Otol Rhinol Laryngol,2003,112(11):915～920.
[37] Thibeault S L, Klemuk S A, Smith M E, et al. In vivo comparison of biomimeticapproaches for tissue regeneration of the scarred vocal fold[J]. Tissue Eng Part A,2009,15(7):1481～1487.
[38] Johnson B Q, Fox R, Chen X, et al. Tissue regeneration of the vocal fold using bonemarrow mesenchymal stem cells and synthetic extracellular matrix injections inrats[J]. Laryngoscope,2010,120(3):537～545.
[39] Kanemaru S, Nakamura T, Yamashita M, et al. Destiny of autologous bonemarrow-derived stromal cells implanted in the vocal fold[J]. Ann Otol RhinolLaryngol,2005,114(12):907～912.
[40] Kumai Y, Kobler J B, Herrera V L, et al. Perspectives on adipose-derivedstem/stromal cells as potential treatment for scarred vocal folds: opportunity andchallenges[J]. Curr Stem Cell Res Ther,2010,5(2):175～181.
[41] Long J L, Zuk P, Berke G S, et al. Epithelial differentiation of adipose-derived stemcells for laryngeal tissue engineering[J]. Laryngoscope,2010,120(1):125～131.
[42] Hertegard S, Cedervall J, Svensson B, et al. Viscoelastic and histologic properties inscarred rabbit vocal folds after mesenchymal stem cell injection[J]. Laryngoscope,2006,116(7):1248～1254.
[43] Svensson B, Nagubothu R S, Cedervall J, et al. Injection of human mesenchymalstem cells improves healing of scarred vocal folds: analysis using a xenograftmodel[J]. Laryngoscope,2010,120(7):1370～1375.
[44] Shin'Oka T, Imai Y, Ikada Y. Transplantation of a tissue-engineered pulmonaryartery[J]. N Engl J Med,2001,344(7):532～533.
[45] Atala A, Bauer S B, Soker S, et al. Tissue-engineered autologous bladders forpatients needing cystoplasty[J]. Lancet,2006,367(9518):1241～1246.
[46] Macchiarini P, Jungebluth P, Go T, et al. Clinical transplantation of atissue-engineered airway[J]. Lancet,2008,372(9655):2023～2030.
[47] Ott H C, Matthiesen T S, Goh S K, et al. Perfusion-decellularized matrix: usingnature's platform to engineer a bioartificial heart[J]. Nat Med,2008,14(2):213～221.
[48] Uygun B E, Soto-Gutierrez A, Yagi H, et al. Organ reengineering throughdevelopment of a transplantable recellularized liver graft using decellularized livermatrix[J]. Nat Med,2010,16(7):814～820.
[49] Petersen T H, Calle E A, Zhao L, et al. Tissue-engineered lungs for in vivoimplantation[J]. Science,2010,329(5991):538～541.
[50] Sellaro T L, Ranade A, Faulk D M, et al. Maintenance of human hepatocyte functionin vitro by liver-derived extracellular matrix gels[J]. Tissue Eng Part A,2010,16(3):1075～1082.
[51] Lin P, Chan W C, Badylak S F, et al. Assessing porcine liver-derived biomatrix forhepatic tissue engineering[J]. Tissue Eng,2004,10(7-8):1046～1053.
[52] Sellaro T L, Ravindra A K, Stolz D B, et al. Maintenance of hepatic sinusoidalendothelial cell phenotype in vitro using organ-specific extracellular matrixscaffolds[J]. Tissue Eng,2007,13(9):2301～2310.
[53] Lin Y M, Zhang A, Rippon H J, et al. Tissue engineering of lung: the effect ofextracellular matrix on the differentiation of embryonic stem cells to pneumocytes[J].Tissue Eng Part A,2010,16(5):1515～1526.
[54] Nelson C M, Bissell M J. Of extracellular matrix, scaffolds, and signaling: tissuearchitecture regulates development, homeostasis, and cancer[J]. Annu Rev CellDev Biol,2006,22:287～309.
[55] Bernard M P, Chu M L, Myers J C, et al. Nucleotide sequences of complementarydeoxyribonucleic acids for the pro alpha1chain of human type I procollagen.Statistical evaluation of structures that are conserved during evolution[J].Biochemistry,1983,22(22):5213～5223.
[56] Exposito J Y, D'Alessio M, Solursh M, et al. Sea urchin collagen evolutionarilyhomologous to vertebrate pro-alpha2(I) collagen[J]. J Biol Chem,1992,267(22):15559～15562.
[57] Badylak S F. The extracellular matrix as a biologic scaffold material[J].Biomaterials,2007,28(25):3587～3593.
[58] Chen R N, Ho H O, Tsai Y T, et al. Process development of an acellular dermalmatrix (ADM) for biomedical applications[J]. Biomaterials,2004,25(13):2679～2686.
[59] Kropp B P, Eppley B L, Prevel C D, et al. Experimental assessment of smallintestinal submucosa as a bladder wall substitute[J]. Urology,1995,46(3):396～400.
[60] Gilbert T W, Stolz D B, Biancaniello F, et al. Production and characterization ofECM powder: implications for tissue engineering applications[J]. Biomaterials,2005,26(12):1431～1435.
[61] Schmidt C E, Baier J M. Acellular vascular tissues: natural biomaterials for tissuerepair and tissue engineering[J]. Biomaterials,2000,21(22):2215～2231.
[62] Grauss R W, Hazekamp M G, Oppenhuizen F, et al. Histological evaluation ofdecellularised porcine aortic valves: matrix changes due to different decellularisationmethods[J]. Eur J Cardiothorac Surg,2005,27(4):566～571.
[63] Gilbert T W, Sellaro T L, Badylak S F. Decellularization of tissues and organs[J].Biomaterials,2006,27(19):3675～3683.
[64] Badylak S F, Freytes D O, Gilbert T W. Extracellular matrix as a biological scaffoldmaterial: Structure and function[J]. Acta Biomater,2009,5(1):1～13.
[65] Crapo P M, Gilbert T W, Badylak S F. An overview of tissue and whole organdecellularization processes[J]. Biomaterials,2011,32(12):3233～3243.
[66] Brown B, Lindberg K, Reing J, et al. The basement membrane component ofbiologic scaffolds derived from extracellular matrix[J]. Tissue Eng,2006,12(3):519～526.
[67] Sacks M S, Gloeckner D C. Quantification of the fiber architecture and biaxialmechanical behavior of porcine intestinal submucosa[J]. J Biomed Mater Res,1999,46(1):1～10.
[68] Gong J, Sagiv O, Cai H, et al. Effects of extracellular matrix and neighboring cellson induction of human embryonic stem cells into retinal or retinal pigment epithelialprogenitors[J]. Exp Eye Res,2008,86(6):957～965.
[69] Nguyen C, Hosokawa T, Kuroiwa M, et al. Differential regulation of theCdk5-dependent phosphorylation sites of inhibitor-1and DARPP-32bydepolarization[J]. J Neurochem,2007,103(4):1582～1593.
[70] Voytik-Harbin S L, Brightman A O, Kraine M R, et al. Identification of extractablegrowth factors from small intestinal submucosa[J]. J Cell Biochem,1997,67(4):478～491.
[71] Hodde J P, Record R D, Liang H A, et al. Vascular endothelial growth factor inporcine-derived extracellular matrix[J]. Endothelium,2001,8(1):11～24.
[72] Hodde J P, Record R D, Tullius R S, et al. Retention of endothelial cell adherence toporcine-derived extracellular matrix after disinfection and sterilization[J]. TissueEng,2002,8(2):225～234.
[73] Brennan E P, Reing J, Chew D, et al. Antibacterial activity within degradationproducts of biological scaffolds composed of extracellular matrix[J]. Tissue Eng,2006,12(10):2949～2955.
[74] Crapo P M, Gilbert T W, Badylak S F. An overview of tissue and whole organdecellularization processes[J]. Biomaterials,2011,32(12):3233～3243.
[75] Keane T J, Londono R, Turner N J, et al. Consequences of ineffectivedecellularization of biologic scaffolds on the host response[J]. Biomaterials,2012,33(6):1771～1781.
[76] Zheng M H, Chen J, Kirilak Y, et al. Porcine small intestine submucosa (SIS) is notan acellular collagenous matrix and contains porcine DNA: possible implications inhuman implantation[J]. J Biomed Mater Res B Appl Biomater,2005,73(1):61～67.
[77] Galili U. The alpha-Gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) inxenotransplantation[J]. Biochimie,2001,83(7):557～563.
[78] Daly A B, Wallis J M, Borg Z D, et al. Initial binding and recellularization ofdecellularized mouse lung scaffolds with bone marrow-derived mesenchymalstromal cells[J]. Tissue Eng Part A,2012,18(1-2):1～16.
[79] Gilbert T W, Wognum S, Joyce E M, et al. Collagen fiber alignment and biaxialmechanical behavior of porcine urinary bladder derived extracellular matrix[J].Biomaterials,2008,29(36):4775～4782.
[80] Hodde J, Janis A, Hiles M. Effects of sterilization on an extracellular matrix scaffold:part II. Bioactivity and matrix interaction[J]. J Mater Sci Mater Med,2007,18(4):545～550.
[81] Dong X, Wei X, Yi W, et al. RGD-modified acellular bovine pericardium as abioprosthetic scaffold for tissue engineering[J]. J Mater Sci Mater Med,2009,20(11):2327～2336.
[82] Reing J E, Brown B N, Daly K A, et al. The effects of processing methods uponmechanical and biologic properties of porcine dermal extracellular matrixscaffolds[J]. Biomaterials,2010,31(33):8626～8633.
[83] Gorschewsky O, Puetz A, Riechert K, et al. Quantitative analysis of biochemicalcharacteristics of bone-patellar tendon-bone allografts[J]. Biomed Mater Eng,2005,15(6):403～411.
[84] Xu C C, Chan R W, Tirunagari N. A biodegradable, acellular xenogeneic scaffoldfor regeneration of the vocal fold lamina propria[J]. Tissue Eng,2007,13(3):551～566.
[85] Cox B, Emili A. Tissue subcellular fractionation and protein extraction for use inmass-spectrometry-based proteomics[J]. Nat Protoc,2006,1(4):1872～1878.
[86] Alhamdani M S, Schroder C, Werner J, et al. Single-step procedure for the isolationof proteins at near-native conditions from mammalian tissue for proteomic analysison antibody microarrays[J]. J Proteome Res,2010,9(2):963～971.
[87] Meyer S R, Chiu B, Churchill T A, et al. Comparison of aortic valve allograftdecellularization techniques in the rat[J]. J Biomed Mater Res A,2006,79(2):254～262.
[88] Nakayama K H, Batchelder C A, Lee C I, et al. Decellularized rhesus monkeykidney as a three-dimensional scaffold for renal tissue engineering[J]. Tissue EngPart A,2010,16(7):2207～2216.
[89] Lumpkins S B, Pierre N, McFetridge P S. A mechanical evaluation of threedecellularization methods in the design of a xenogeneic scaffold for tissueengineering the temporomandibular joint disc[J]. Acta Biomater,2008,4(4):808～816.
[90] Ott H C, Clippinger B, Conrad C, et al. Regeneration and orthotopic transplantationof a bioartificial lung[J]. Nat Med,2010,16(8):927～933.
[91] Kasimir M T, Rieder E, Seebacher G, et al. Comparison of differentdecellularization procedures of porcine heart valves[J]. Int J Artif Organs,2003,26(5):421～427.
[92] Woods T, Gratzer P F. Effectiveness of three extraction techniques in thedevelopment of a decellularized bone-anterior cruciate ligament-bone graft[J].Biomaterials,2005,26(35):7339～7349.
[93] Funamoto S, Nam K, Kimura T, et al. The use of high-hydrostatic pressuretreatment to decellularize blood vessels[J]. Biomaterials,2010,31(13):3590～3595.
[94] Cebotari S, Tudorache I, Jaekel T, et al. Detergent decellularization of heart valvesfor tissue engineering: toxicological effects of residual detergents on humanendothelial cells[J]. Artif Organs,2010,34(3):206～210.
[95] Gui L, Chan S A, Breuer C K, et al. Novel utilization of serum in tissuedecellularization[J]. Tissue Eng Part C Methods,2010,16(2):173～184.
[96] Hudson T W, Liu S Y, Schmidt C E. Engineering an improved acellular nerve graftvia optimized chemical processing[J]. Tissue Eng,2004,10(9-10):1346～1358.
[97] Cebotari S, Tudorache I, Schilling T, et al.[Heart valve and myocardial tissueengineering][J]. Herz,2010,35(5):334～341.
[98] Prasertsung I, Kanokpanont S, Bunaprasert T, et al. Development of acellular dermisfrom porcine skin using periodic pressurized technique[J]. J Biomed Mater Res BAppl Biomater,2008,85(1):210～219.
[99] Levy R J, Vyavahare N, Ogle M, et al. Inhibition of cusp and aortic wallcalcification in ethanol-and aluminum-treated bioprosthetic heart valves in sheep:background, mechanisms, and synergism[J]. J Heart Valve Dis,2003,12(2):209～216,216.
[100] Brown B N, Barnes C A, Kasick R T, et al. Surface characterization of extracellularmatrix scaffolds[J]. Biomaterials,2010,31(3):428～437.
[101] Montoya C V, McFetridge P S. Preparation of ex vivo-based biomaterials usingconvective flow decellularization[J]. Tissue Eng Part C Methods,2009,15(2):191～200.
[102] Jamur M C, Oliver C. Cell fixatives for immunostaining[J]. Methods Mol Biol,2010,588:55～61.
[103] Horowitz B, Bonomo R, Prince A M, et al. Solvent/detergent-treated plasma: avirus-inactivated substitute for fresh frozen plasma[J]. Blood,1992,79(3):826～831.
[104] Deeken C R, White A K, Bachman S L, et al. Method of preparing a decellularizedporcine tendon using tributyl phosphate[J]. J Biomed Mater Res B Appl Biomater,2011,96(2):199～206.
[105] Cartmell J S, Dunn M G. Development of cell-seeded patellar tendon allografts foranterior cruciate ligament reconstruction[J]. Tissue Eng,2004,10(7-8):1065～1075.
[106] Waldrop F S, Puchtler H, Meloan S N, et al. Histochemical investigations ofdifferent types of collagen[J]. Acta Histochem Suppl,1980,21:23～31.
[107] Yang M, Chen C Z, Wang X N, et al. Favorable effects of the detergent and enzymeextraction method for preparing decellularized bovine pericardium scaffold fortissue engineered heart valves[J]. J Biomed Mater Res B Appl Biomater,2009,91(1):354～361.
[108] Schenke-Layland K, Vasilevski O, Opitz F, et al. Impact of decellularization ofxenogeneic tissue on extracellular matrix integrity for tissue engineering of heartvalves[J]. J Struct Biol,2003,143(3):201～208.
[109] Brown B N, Freund J M, Han L, et al. Comparison of three methods for thederivation of a biologic scaffold composed of adipose tissue extracellular matrix[J].Tissue Eng Part C Methods,2011,17(4):411～421.
[110] Xu H, Wan H, Sandor M, et al. Host response to human acellular dermal matrixtransplantation in a primate model of abdominal wall repair[J]. Tissue Eng Part A,2008,14(12):2009～2019.
[111] Klebe R J. Isolation of a collagen-dependent cell attachment factor[J]. Nature,1974,250(463):248～251.
[112] Gillies A R, Smith L R, Lieber R L, et al. Method for decellularizing skeletal musclewithout detergents or proteolytic enzymes[J]. Tissue Eng Part C Methods,2011,17(4):383～389.
[113] Lehr E J, Rayat G R, Chiu B, et al. Decellularization reduces immunogenicity ofsheep pulmonary artery vascular patches[J]. J Thorac Cardiovasc Surg,2011,141(4):1056～1062.
[114] Hopkinson A, Shanmuganathan V A, Gray T, et al. Optimization of amnioticmembrane (AM) denuding for tissue engineering[J]. Tissue Eng Part C Methods,2008,14(4):371～381.
[115] Hashimoto Y, Funamoto S, Sasaki S, et al. Preparation and characterization ofdecellularized cornea using high-hydrostatic pressurization for corneal tissueengineering[J]. Biomaterials,2010,31(14):3941～3948.
[116] Lee R C. Cell injury by electric forces[J]. Ann N Y Acad Sci,2005,1066:85～91.
[117] Phillips M, Maor E, Rubinsky B. Nonthermal irreversible electroporation for tissuedecellularization[J]. J Biomech Eng,2010,132(9):91003.
[118] Sano M B, Neal R N, Garcia P A, et al. Towards the creation of decellularized organconstructs using irreversible electroporation and active mechanical perfusion[J].Biomed Eng Online,2010,9:83.
[119] Henderson P W, Nagineni V V, Harper A, et al. Development of an acellularbioengineered matrix with a dominant vascular pedicle[J]. J Surg Res,2010,164(1):1～5.
[120] Wainwright J M, Czajka C A, Patel U B, et al. Preparation of cardiac extracellularmatrix from an intact porcine heart[J]. Tissue Eng Part C Methods,2010,16(3):525～532.
[121] Price A P, England K A, Matson A M, et al. Development of a decellularized lungbioreactor system for bioengineering the lung: the matrix reloaded[J]. Tissue EngPart A,2010,16(8):2581～2591.
[122] Cortiella J, Niles J, Cantu A, et al. Influence of acellular natural lung matrix onmurine embryonic stem cell differentiation and tissue formation[J]. Tissue Eng PartA,2010,16(8):2565～2580.
[123] Shupe T, Williams M, Brown A, et al. Method for the decellularization of intact ratliver[J]. Organogenesis,2010,6(2):134～136.
[124] Ross E A, Williams M J, Hamazaki T, et al. Embryonic stem cells proliferate anddifferentiate when seeded into kidney scaffolds[J]. J Am Soc Nephrol,2009,20(11):2338～2347.
[125] Marolt D, Knezevic M, Novakovic G V. Bone tissue engineering with human stemcells[J]. Stem Cell Res Ther,2010,1(2):10.
[126] Kofidis T, de Bruin J L, Hoyt G, et al. Myocardial restoration with embryonic stemcell bioartificial tissue transplantation[J]. J Heart Lung Transplant,2005,24(6):737～744.
[127] Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells fromadult human fibroblasts by defined factors[J]. Cell,2007,131(5):861～872.
[128] Zhang Y, Shen W, Sun B, et al. Plasticity of marrow mesenchymal stem cells fromhuman first-trimester fetus: from single-cell clone to neuronal differentiation[J]. CellReprogram,2011,13(1):57～64.
[129] Decembrini S, Cananzi M, Gualdoni S, et al. Comparative analysis of the retinalpotential of embryonic stem cells and amniotic fluid-derived stem cells[J]. StemCells Dev,2011,20(5):851～863.
[130] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouseembryonic and adult fibroblast cultures by defined factors[J]. Cell,2006,126(4):663～676.
[131] Prasad V K, Kurtzberg J. Transplant outcomes in mucopolysaccharidoses[J]. SeminHematol,2010,47(1):59～69.
[132] Shen Q, Goderie S K, Jin L, et al. Endothelial cells stimulate self-renewal andexpand neurogenesis of neural stem cells[J]. Science,2004,304(5675):1338～1340.
[133] Menon N G, Rodriguez E D, Byrnes C K, et al. Revascularization of humanacellular dermis in full-thickness abdominal wall reconstruction in the rabbitmodel[J]. Ann Plast Surg,2003,50(5):523～527.
[134] Deutsch M, Meinhart J, Vesely M, et al. In vitro endothelialization of expandedpolytetrafluoroethylene grafts: a clinical case report after41months ofimplantation[J]. J Vasc Surg,1997,25(4):757～763.
[135] Lee C Y, Mangino M J. Preservation methods for kidney and liver[J].Organogenesis,2009,5(3):105～112.
[136] Niklason L E, Gao J, Abbott W M, et al. Functional arteries grown in vitro[J].Science,1999,284(5413):489～493.
[137] Park J, Li Y, Berthiaume F, et al. Radial flow hepatocyte bioreactor using stackedmicrofabricated grooved substrates[J]. Biotechnol Bioeng,2008,99(2):455～467.
[138] Ratcliffe A, Niklason L E. Bioreactors and bioprocessing for tissue engineering[J].Ann N Y Acad Sci,2002,961:210～215.
[139] Nahmias Y, Kramvis Y, Barbe L, et al. A novel formulation of oxygen-carryingmatrix enhances liver-specific function of cultured hepatocytes[J]. FASEB J,2006,20(14):2531～2533.
[140] Lovett M, Lee K, Edwards A, et al. Vascularization strategies for tissueengineering[J]. Tissue Eng Part B Rev,2009,15(3):353～370.
[141] Sewnaik A, Hoorweg J J, Knegt P P, et al. Treatment of hypopharyngeal carcinoma:analysis of nationwide study in the Netherlands over a10-year period[J]. ClinOtolaryngol,2005,30(1):52～57.
[142] Keereweer S, de Wilt J H, Sewnaik A, et al. Early and long-term morbidity aftertotal laryngopharyngectomy[J]. Eur Arch Otorhinolaryngol,2010,267(9):1437～1444.
[143] Ramirez M J, Ferriol E E, Domenech F G, et al. Psychosocial adjustment in patientssurgically treated for laryngeal cancer[J]. Otolaryngol Head Neck Surg,2003,129(1):92～97.
[144] Baiguera S, Gonfiotti A, Jaus M, et al. Development of bioengineered humanlarynx[J]. Biomaterials,2011,32(19):4433～4442.
[145] Haykal S, Soleas J P, Salna M, et al. Evaluation of the structural integrity andextracellular matrix components of tracheal allografts following cyclicaldecellularization techniques: comparison of three protocols[J]. Tissue Eng Part CMethods,2012,18(8):614～623.
[146] Bhosale A M, Richardson J B. Articular cartilage: structure, injuries and review ofmanagement[J]. Br Med Bull,2008,87:77～95.
[147] Liu Y, Nakamura T, Yamamoto Y, et al. Immunosuppressant-freeallotransplantation of the trachea: the antigenicity of tracheal grafts can be reducedby removing the epithelium and mixed glands from the graft by detergenttreatment[J]. J Thorac Cardiovasc Surg,2000,120(1):108～114.
[148] Huey D J, Sanchez-Adams J, Willard V P, et al. Immunogenicity of bovine andleporine articular chondrocytes and meniscus cells[J]. Tissue Eng Part A,2012,18(5-6):568～575.
[149] Bujia J, Wilmes E, Hammer C, et al. Class II antigenicity of human cartilage:relevance to the use of homologous cartilage graft for reconstructive surgery[J]. AnnPlast Surg,1991,26(6):541～543.
[150] Langer F, Gross A E. Immunogenicity of allograft articular cartilage[J]. J Bone JointSurg Am,1974,56(2):297～304.
[151]马瑞娜,崔鹏程.喉移植免疫学研究进展[J].听力学及言语疾病杂志,2010,18(3):304～306.
[152] Rees L E, Ayoub O, Haverson K, et al. Differential major histocompatibilitycomplex class II locus expression on human laryngeal epithelium[J]. Clin ExpImmunol,2003,134(3):497～502.
[153] Wang E C, Damrose E J, Mendelsohn A H, et al. Distribution of class I and IIhuman leukocyte antigens in the larynx[J]. Otolaryngol Head Neck Surg,2006,134(2):280～287.
[154] Pittenger M F, Mackay A M, Beck S C, et al. Multilineage potential of adult humanmesenchymal stem cells[J]. Science,1999,284(5411):143～147.
[155] Wilson A S, Hobbs B G, Shen W Y, et al. Argon laser photocoagulation-inducedmodification of gene expression in the retina[J]. Invest Ophthalmol Vis Sci,2003,44(4):1426～1434.
[156] Otto A, Collins-Hooper H, Patel K. The origin, molecular regulation and therapeuticpotential of myogenic stem cell populations[J]. J Anat,2009,215(5):477～497.
[157] Ferrari G, Cusella-De A G, Coletta M, et al. Muscle regeneration by bonemarrow-derived myogenic progenitors[J]. Science,1998,279(5356):1528～1530.
[158] Noiseux N, Gnecchi M, Lopez-Ilasaca M, et al. Mesenchymal stem cellsoverexpressing Akt dramatically repair infarcted myocardium and improve cardiacfunction despite infrequent cellular fusion or differentiation[J]. Mol Ther,2006,14(6):840～850.
[159] Baraniak P R, McDevitt T C. Stem cell paracrine actions and tissue regeneration[J].Regen Med,2010,5(1):121～143.
[160]李秋果.组织工程支架材料研究进展及发展前景[J].长江大学学报自然科学版：农学卷,2012,9(2):21～25.
[161]侯楠,崔鹏程,罗家胜,等.灌注法与浸泡法构建去细胞全喉支架的对比研究[J].中国修复重建外科杂志,2009,(5):623～626.
[162]许莉,崔鹏程,陈志峰,等.犬去细胞喉支架的生物力学研究[J].中华耳鼻咽喉头颈外科杂志,2011,46(4):331～335.
[163]马瑞娜,崔鹏程,侯楠,等.去细胞全喉软骨支架的免疫学研究[J].中华耳鼻咽喉头颈外科杂志,2010,45(3):233～237.
[2] Minnich D J, Mathisen D J. Anatomy of the trachea, carina, and bronchi[J]. ThoracSurg Clin,2007,17(4):571～585.
[3] Macchiarini P, Jungebluth P, Go T, et al. Clinical transplantation of atissue-engineered airway[J]. Lancet,2008,372(9655):2023～2030.
[4] Yamashita M, Hirano S, Kanemaru S, et al. Side population cells in the human vocalfold[J]. Ann Otol Rhinol Laryngol,2007,116(11):847～852.
[5] Goodell M A, Brose K, Paradis G, et al. Isolation and functional properties ofmurine hematopoietic stem cells that are replicating in vivo[J]. J Exp Med,1996,183(4):1797～1806.
[6] Gugatschka M, Kojima T, Ohno S, et al. Recruitment patterns of side populationcells during wound healing in rat vocal folds[J]. Laryngoscope,2011,121(8):1662～1667.
[7] Hanson S E, Kim J, Johnson B H, et al. Characterization of mesenchymal stem cellsfrom human vocal fold fibroblasts[J]. Laryngoscope,2010,120(3):546～551.
[8] Gugatschka M, Ohno S, Saxena A, et al. Regenerative medicine of the larynx.Where are we today? A review[J]. J Voice,2012,26(5):670～677.
[9] Hirano S, Kishimoto Y, Suehiro A, et al. Regeneration of Aged Vocal Fold: FirstHuman Case Treated With Fibroblast Growth Factor[J]. The Laryngoscope,2008,118(12):2254～2259.
[10] Hirano S, Kishimoto Y, Suehiro A, et al. Regeneration of aged vocal fold: Firsthuman case treated with fibroblast growth factor[J]. The Laryngoscope,2009,119(1):197～202.
[11] Hirano S, Nagai H, Tateya I, et al. Regeneration of aged vocal folds with basicfibroblast growth factor in a rat model: a preliminary report[J]. Ann Otol RhinolLaryngol,2005,114(4):304～308.
[12] Ohno T, Hirano S, Rousseau B. Gene expression of transforming growthfactor-beta1and hepatocyte growth factor during wound healing of injured ratvocal fold[J]. Laryngoscope,2009,119(4):806～810.
[13] Ohno T, Yoo M J, Swanson E R, et al. Regenerative effects of basic fibroblastgrowth factor on extracellular matrix production in aged rat vocal folds[J]. Ann OtolRhinol Laryngol,2009,118(8):559～564.
[14] Matsumoto K, Nakamura T. Hepatocyte growth factor (HGF) as a tissue organizerfor organogenesis and regeneration[J]. Biochem Biophys Res Commun,1997,239(3):639～644.
[15] Hirano S, Bless D, Heisey D, et al. Roles of hepatocyte growth factor andtransforming growth factor beta1in production of extracellular matrix by caninevocal fold fibroblasts[J]. Laryngoscope,2003,113(1):144～148.
[16] Hirano S, Bless D M, Nagai H, et al. Growth factor therapy for vocal fold scarring ina canine model[J]. Ann Otol Rhinol Laryngol,2004,113(10):777～785.
[17] Ohno T, Hirano S, Kanemaru S, et al. Drug delivery system of hepatocyte growthfactor for the treatment of vocal fold scarring in a canine model[J]. Ann OtolRhinol Laryngol,2007,116(10):762～769.
[18] Sheen-Chen S M, Liu Y W, Eng H L, et al. Serum levels of hepatocyte growthfactor in patients with breast cancer[J]. Cancer Epidemiol Biomarkers Prev,2005,14(3):715～717.
[19] Farran A J, Teller S S, Jha A K, et al. Effects of matrix composition, microstructure,and viscoelasticity on the behaviors of vocal fold fibroblasts cultured inthree-dimensional hydrogel networks[J]. Tissue Eng Part A,2010,16(4):1247～1261.
[20] Butler J E, Hammond T H, Gray S D. Gender-related differences of hyaluronic aciddistribution in the human vocal fold[J]. Laryngoscope,2001,111(5):907～911.
[21] Munoz-Pinto D J, Jimenez-Vergara A C, Gelves L M, et al. Probing vocal foldfibroblast response to hyaluronan in3D contexts[J]. Biotechnol Bioeng,2009,104(4):821～831.
[22] Luo Y, Kobler J B, Zeitels S M, et al. Effects of growth factors on extracellularmatrix production by vocal fold fibroblasts in3-dimensional culture[J]. Tissue Eng,2006,12(12):3365～3374.
[23] Park H, Karajanagi S, Wolak K, et al. Three-dimensional hydrogel model usingadipose-derived stem cells for vocal fold augmentation[J]. Tissue Eng Part A,2010,16(2):535～543.
[24] Luo Y, Kobler J B, Heaton J T, et al. Injectable hyaluronic acid-dextran hydrogelsand effects of implantation in ferret vocal fold[J]. J Biomed Mater Res B ApplBiomater,2010,93(2):386～393.
[25] Webb K, Hitchcock R W, Smeal R M, et al. Cyclic strain increases fibroblastproliferation, matrix accumulation, and elastic modulus of fibroblast-seededpolyurethane constructs[J]. J Biomech,2006,39(6):1136～1144.
[26] Webb K, Li W, Hitchcock R W, et al. Comparison of human fibroblast ECM-relatedgene expression on elastic three-dimensional substrates relative to two-dimensionalfilms of the same material[J]. Biomaterials,2003,24(25):4681～4690.
[27] Thibeault S L, Duflo S. Inflammatory cytokine responses to synthetic extracellularmatrix injection to the vocal fold lamina propria[J]. Ann Otol Rhinol Laryngol,2008,117(3):221～226.
[28] Hahn M S, Teply B A, Stevens M M, et al. Collagen composite hydrogels for vocalfold lamina propria restoration[J]. Biomaterials,2006,27(7):1104～1109.
[29] Huber J E, Spievack A, Simmons-Byrd A, et al. Extracellular matrix as a scaffoldfor laryngeal reconstruction[J]. Ann Otol Rhinol Laryngol,2003,112(5):428～433.
[30] Xu C C, Chan R W. Pore architecture of a bovine acellular vocal fold scaffold[J].Tissue Eng Part A,2008,14(11):1893～1903.
[31] Xu C C, Chan R W, Weinberger D G, et al. A bovine acellular scaffold for vocalfold reconstruction in a rat model[J]. J Biomed Mater Res A,2010,92(1):18～32.
[32] Chan R W, Rodriguez M L, McFetridge P S. The human umbilical vein withWharton's jelly as an allogeneic, acellular construct for vocal fold restoration[J].Tissue Eng Part A,2009,15(11):3537～3546.
[33] Wei H J, Liang H C, Lee M H, et al. Construction of varying porous structures inacellular bovine pericardia as a tissue-engineering extracellular matrix[J].Biomaterials,2005,26(14):1905～1913.
[34] Xu C C, Chan R W, Weinberger D G, et al. Controlled release of hepatocyte growthfactor from a bovine acellular scaffold for vocal fold reconstruction[J]. J BiomedMater Res A,2010,93(4):1335～1347.
[35] Chhetri D K, Head C, Revazova E, et al. Lamina propria replacement therapy withcultured autologous fibroblasts for vocal fold scars[J]. Otolaryngol Head Neck Surg,2004,131(6):864～870.
[36] Kanemaru S, Nakamura T, Omori K, et al. Regeneration of the vocal fold usingautologous mesenchymal stem cells[J]. Ann Otol Rhinol Laryngol,2003,112(11):915～920.
[37] Thibeault S L, Klemuk S A, Smith M E, et al. In vivo comparison of biomimeticapproaches for tissue regeneration of the scarred vocal fold[J]. Tissue Eng Part A,2009,15(7):1481～1487.
[38] Johnson B Q, Fox R, Chen X, et al. Tissue regeneration of the vocal fold using bonemarrow mesenchymal stem cells and synthetic extracellular matrix injections inrats[J]. Laryngoscope,2010,120(3):537～545.
[39] Kanemaru S, Nakamura T, Yamashita M, et al. Destiny of autologous bonemarrow-derived stromal cells implanted in the vocal fold[J]. Ann Otol RhinolLaryngol,2005,114(12):907～912.
[40] Kumai Y, Kobler J B, Herrera V L, et al. Perspectives on adipose-derivedstem/stromal cells as potential treatment for scarred vocal folds: opportunity andchallenges[J]. Curr Stem Cell Res Ther,2010,5(2):175～181.
[41] Long J L, Zuk P, Berke G S, et al. Epithelial differentiation of adipose-derived stemcells for laryngeal tissue engineering[J]. Laryngoscope,2010,120(1):125～131.
[42] Hertegard S, Cedervall J, Svensson B, et al. Viscoelastic and histologic properties inscarred rabbit vocal folds after mesenchymal stem cell injection[J]. Laryngoscope,2006,116(7):1248～1254.
[43] Svensson B, Nagubothu R S, Cedervall J, et al. Injection of human mesenchymalstem cells improves healing of scarred vocal folds: analysis using a xenograftmodel[J]. Laryngoscope,2010,120(7):1370～1375.
[44] Shin'Oka T, Imai Y, Ikada Y. Transplantation of a tissue-engineered pulmonaryartery[J]. N Engl J Med,2001,344(7):532～533.
[45] Atala A, Bauer S B, Soker S, et al. Tissue-engineered autologous bladders forpatients needing cystoplasty[J]. Lancet,2006,367(9518):1241～1246.
[46] Macchiarini P, Jungebluth P, Go T, et al. Clinical transplantation of atissue-engineered airway[J]. Lancet,2008,372(9655):2023～2030.
[47] Ott H C, Matthiesen T S, Goh S K, et al. Perfusion-decellularized matrix: usingnature's platform to engineer a bioartificial heart[J]. Nat Med,2008,14(2):213～221.
[48] Uygun B E, Soto-Gutierrez A, Yagi H, et al. Organ reengineering throughdevelopment of a transplantable recellularized liver graft using decellularized livermatrix[J]. Nat Med,2010,16(7):814～820.
[49] Petersen T H, Calle E A, Zhao L, et al. Tissue-engineered lungs for in vivoimplantation[J]. Science,2010,329(5991):538～541.
[50] Sellaro T L, Ranade A, Faulk D M, et al. Maintenance of human hepatocyte functionin vitro by liver-derived extracellular matrix gels[J]. Tissue Eng Part A,2010,16(3):1075～1082.
[51] Lin P, Chan W C, Badylak S F, et al. Assessing porcine liver-derived biomatrix forhepatic tissue engineering[J]. Tissue Eng,2004,10(7-8):1046～1053.
[52] Sellaro T L, Ravindra A K, Stolz D B, et al. Maintenance of hepatic sinusoidalendothelial cell phenotype in vitro using organ-specific extracellular matrixscaffolds[J]. Tissue Eng,2007,13(9):2301～2310.
[53] Lin Y M, Zhang A, Rippon H J, et al. Tissue engineering of lung: the effect ofextracellular matrix on the differentiation of embryonic stem cells to pneumocytes[J].Tissue Eng Part A,2010,16(5):1515～1526.
[54] Nelson C M, Bissell M J. Of extracellular matrix, scaffolds, and signaling: tissuearchitecture regulates development, homeostasis, and cancer[J]. Annu Rev CellDev Biol,2006,22:287～309.
[55] Bernard M P, Chu M L, Myers J C, et al. Nucleotide sequences of complementarydeoxyribonucleic acids for the pro alpha1chain of human type I procollagen.Statistical evaluation of structures that are conserved during evolution[J].Biochemistry,1983,22(22):5213～5223.
[56] Exposito J Y, D'Alessio M, Solursh M, et al. Sea urchin collagen evolutionarilyhomologous to vertebrate pro-alpha2(I) collagen[J]. J Biol Chem,1992,267(22):15559～15562.
[57] Badylak S F. The extracellular matrix as a biologic scaffold material[J].Biomaterials,2007,28(25):3587～3593.
[58] Chen R N, Ho H O, Tsai Y T, et al. Process development of an acellular dermalmatrix (ADM) for biomedical applications[J]. Biomaterials,2004,25(13):2679～2686.
[59] Kropp B P, Eppley B L, Prevel C D, et al. Experimental assessment of smallintestinal submucosa as a bladder wall substitute[J]. Urology,1995,46(3):396～400.
[60] Gilbert T W, Stolz D B, Biancaniello F, et al. Production and characterization ofECM powder: implications for tissue engineering applications[J]. Biomaterials,2005,26(12):1431～1435.
[61] Schmidt C E, Baier J M. Acellular vascular tissues: natural biomaterials for tissuerepair and tissue engineering[J]. Biomaterials,2000,21(22):2215～2231.
[62] Grauss R W, Hazekamp M G, Oppenhuizen F, et al. Histological evaluation ofdecellularised porcine aortic valves: matrix changes due to different decellularisationmethods[J]. Eur J Cardiothorac Surg,2005,27(4):566～571.
[63] Gilbert T W, Sellaro T L, Badylak S F. Decellularization of tissues and organs[J].Biomaterials,2006,27(19):3675～3683.
[64] Badylak S F, Freytes D O, Gilbert T W. Extracellular matrix as a biological scaffoldmaterial: Structure and function[J]. Acta Biomater,2009,5(1):1～13.
[65] Crapo P M, Gilbert T W, Badylak S F. An overview of tissue and whole organdecellularization processes[J]. Biomaterials,2011,32(12):3233～3243.
[66] Brown B, Lindberg K, Reing J, et al. The basement membrane component ofbiologic scaffolds derived from extracellular matrix[J]. Tissue Eng,2006,12(3):519～526.
[67] Sacks M S, Gloeckner D C. Quantification of the fiber architecture and biaxialmechanical behavior of porcine intestinal submucosa[J]. J Biomed Mater Res,1999,46(1):1～10.
[68] Gong J, Sagiv O, Cai H, et al. Effects of extracellular matrix and neighboring cellson induction of human embryonic stem cells into retinal or retinal pigment epithelialprogenitors[J]. Exp Eye Res,2008,86(6):957～965.
[69] Nguyen C, Hosokawa T, Kuroiwa M, et al. Differential regulation of theCdk5-dependent phosphorylation sites of inhibitor-1and DARPP-32bydepolarization[J]. J Neurochem,2007,103(4):1582～1593.
[70] Voytik-Harbin S L, Brightman A O, Kraine M R, et al. Identification of extractablegrowth factors from small intestinal submucosa[J]. J Cell Biochem,1997,67(4):478～491.
[71] Hodde J P, Record R D, Liang H A, et al. Vascular endothelial growth factor inporcine-derived extracellular matrix[J]. Endothelium,2001,8(1):11～24.
[72] Hodde J P, Record R D, Tullius R S, et al. Retention of endothelial cell adherence toporcine-derived extracellular matrix after disinfection and sterilization[J]. TissueEng,2002,8(2):225～234.
[73] Brennan E P, Reing J, Chew D, et al. Antibacterial activity within degradationproducts of biological scaffolds composed of extracellular matrix[J]. Tissue Eng,2006,12(10):2949～2955.
[74] Crapo P M, Gilbert T W, Badylak S F. An overview of tissue and whole organdecellularization processes[J]. Biomaterials,2011,32(12):3233～3243.
[75] Keane T J, Londono R, Turner N J, et al. Consequences of ineffectivedecellularization of biologic scaffolds on the host response[J]. Biomaterials,2012,33(6):1771～1781.
[76] Zheng M H, Chen J, Kirilak Y, et al. Porcine small intestine submucosa (SIS) is notan acellular collagenous matrix and contains porcine DNA: possible implications inhuman implantation[J]. J Biomed Mater Res B Appl Biomater,2005,73(1):61～67.
[77] Galili U. The alpha-Gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) inxenotransplantation[J]. Biochimie,2001,83(7):557～563.
[78] Daly A B, Wallis J M, Borg Z D, et al. Initial binding and recellularization ofdecellularized mouse lung scaffolds with bone marrow-derived mesenchymalstromal cells[J]. Tissue Eng Part A,2012,18(1-2):1～16.
[79] Gilbert T W, Wognum S, Joyce E M, et al. Collagen fiber alignment and biaxialmechanical behavior of porcine urinary bladder derived extracellular matrix[J].Biomaterials,2008,29(36):4775～4782.
[80] Hodde J, Janis A, Hiles M. Effects of sterilization on an extracellular matrix scaffold:part II. Bioactivity and matrix interaction[J]. J Mater Sci Mater Med,2007,18(4):545～550.
[81] Dong X, Wei X, Yi W, et al. RGD-modified acellular bovine pericardium as abioprosthetic scaffold for tissue engineering[J]. J Mater Sci Mater Med,2009,20(11):2327～2336.
[82] Reing J E, Brown B N, Daly K A, et al. The effects of processing methods uponmechanical and biologic properties of porcine dermal extracellular matrixscaffolds[J]. Biomaterials,2010,31(33):8626～8633.
[83] Gorschewsky O, Puetz A, Riechert K, et al. Quantitative analysis of biochemicalcharacteristics of bone-patellar tendon-bone allografts[J]. Biomed Mater Eng,2005,15(6):403～411.
[84] Xu C C, Chan R W, Tirunagari N. A biodegradable, acellular xenogeneic scaffoldfor regeneration of the vocal fold lamina propria[J]. Tissue Eng,2007,13(3):551～566.
[85] Cox B, Emili A. Tissue subcellular fractionation and protein extraction for use inmass-spectrometry-based proteomics[J]. Nat Protoc,2006,1(4):1872～1878.
[86] Alhamdani M S, Schroder C, Werner J, et al. Single-step procedure for the isolationof proteins at near-native conditions from mammalian tissue for proteomic analysison antibody microarrays[J]. J Proteome Res,2010,9(2):963～971.
[87] Meyer S R, Chiu B, Churchill T A, et al. Comparison of aortic valve allograftdecellularization techniques in the rat[J]. J Biomed Mater Res A,2006,79(2):254～262.
[88] Nakayama K H, Batchelder C A, Lee C I, et al. Decellularized rhesus monkeykidney as a three-dimensional scaffold for renal tissue engineering[J]. Tissue EngPart A,2010,16(7):2207～2216.
[89] Lumpkins S B, Pierre N, McFetridge P S. A mechanical evaluation of threedecellularization methods in the design of a xenogeneic scaffold for tissueengineering the temporomandibular joint disc[J]. Acta Biomater,2008,4(4):808～816.
[90] Ott H C, Clippinger B, Conrad C, et al. Regeneration and orthotopic transplantationof a bioartificial lung[J]. Nat Med,2010,16(8):927～933.
[91] Kasimir M T, Rieder E, Seebacher G, et al. Comparison of differentdecellularization procedures of porcine heart valves[J]. Int J Artif Organs,2003,26(5):421～427.
[92] Woods T, Gratzer P F. Effectiveness of three extraction techniques in thedevelopment of a decellularized bone-anterior cruciate ligament-bone graft[J].Biomaterials,2005,26(35):7339～7349.
[93] Funamoto S, Nam K, Kimura T, et al. The use of high-hydrostatic pressuretreatment to decellularize blood vessels[J]. Biomaterials,2010,31(13):3590～3595.
[94] Cebotari S, Tudorache I, Jaekel T, et al. Detergent decellularization of heart valvesfor tissue engineering: toxicological effects of residual detergents on humanendothelial cells[J]. Artif Organs,2010,34(3):206～210.
[95] Gui L, Chan S A, Breuer C K, et al. Novel utilization of serum in tissuedecellularization[J]. Tissue Eng Part C Methods,2010,16(2):173～184.
[96] Hudson T W, Liu S Y, Schmidt C E. Engineering an improved acellular nerve graftvia optimized chemical processing[J]. Tissue Eng,2004,10(9-10):1346～1358.
[97] Cebotari S, Tudorache I, Schilling T, et al.[Heart valve and myocardial tissueengineering][J]. Herz,2010,35(5):334～341.
[98] Prasertsung I, Kanokpanont S, Bunaprasert T, et al. Development of acellular dermisfrom porcine skin using periodic pressurized technique[J]. J Biomed Mater Res BAppl Biomater,2008,85(1):210～219.
[99] Levy R J, Vyavahare N, Ogle M, et al. Inhibition of cusp and aortic wallcalcification in ethanol-and aluminum-treated bioprosthetic heart valves in sheep:background, mechanisms, and synergism[J]. J Heart Valve Dis,2003,12(2):209～216,216.
[100] Brown B N, Barnes C A, Kasick R T, et al. Surface characterization of extracellularmatrix scaffolds[J]. Biomaterials,2010,31(3):428～437.
[101] Montoya C V, McFetridge P S. Preparation of ex vivo-based biomaterials usingconvective flow decellularization[J]. Tissue Eng Part C Methods,2009,15(2):191～200.
[102] Jamur M C, Oliver C. Cell fixatives for immunostaining[J]. Methods Mol Biol,2010,588:55～61.
[103] Horowitz B, Bonomo R, Prince A M, et al. Solvent/detergent-treated plasma: avirus-inactivated substitute for fresh frozen plasma[J]. Blood,1992,79(3):826～831.
[104] Deeken C R, White A K, Bachman S L, et al. Method of preparing a decellularizedporcine tendon using tributyl phosphate[J]. J Biomed Mater Res B Appl Biomater,2011,96(2):199～206.
[105] Cartmell J S, Dunn M G. Development of cell-seeded patellar tendon allografts foranterior cruciate ligament reconstruction[J]. Tissue Eng,2004,10(7-8):1065～1075.
[106] Waldrop F S, Puchtler H, Meloan S N, et al. Histochemical investigations ofdifferent types of collagen[J]. Acta Histochem Suppl,1980,21:23～31.
[107] Yang M, Chen C Z, Wang X N, et al. Favorable effects of the detergent and enzymeextraction method for preparing decellularized bovine pericardium scaffold fortissue engineered heart valves[J]. J Biomed Mater Res B Appl Biomater,2009,91(1):354～361.
[108] Schenke-Layland K, Vasilevski O, Opitz F, et al. Impact of decellularization ofxenogeneic tissue on extracellular matrix integrity for tissue engineering of heartvalves[J]. J Struct Biol,2003,143(3):201～208.
[109] Brown B N, Freund J M, Han L, et al. Comparison of three methods for thederivation of a biologic scaffold composed of adipose tissue extracellular matrix[J].Tissue Eng Part C Methods,2011,17(4):411～421.
[110] Xu H, Wan H, Sandor M, et al. Host response to human acellular dermal matrixtransplantation in a primate model of abdominal wall repair[J]. Tissue Eng Part A,2008,14(12):2009～2019.
[111] Klebe R J. Isolation of a collagen-dependent cell attachment factor[J]. Nature,1974,250(463):248～251.
[112] Gillies A R, Smith L R, Lieber R L, et al. Method for decellularizing skeletal musclewithout detergents or proteolytic enzymes[J]. Tissue Eng Part C Methods,2011,17(4):383～389.
[113] Lehr E J, Rayat G R, Chiu B, et al. Decellularization reduces immunogenicity ofsheep pulmonary artery vascular patches[J]. J Thorac Cardiovasc Surg,2011,141(4):1056～1062.
[114] Hopkinson A, Shanmuganathan V A, Gray T, et al. Optimization of amnioticmembrane (AM) denuding for tissue engineering[J]. Tissue Eng Part C Methods,2008,14(4):371～381.
[115] Hashimoto Y, Funamoto S, Sasaki S, et al. Preparation and characterization ofdecellularized cornea using high-hydrostatic pressurization for corneal tissueengineering[J]. Biomaterials,2010,31(14):3941～3948.
[116] Lee R C. Cell injury by electric forces[J]. Ann N Y Acad Sci,2005,1066:85～91.
[117] Phillips M, Maor E, Rubinsky B. Nonthermal irreversible electroporation for tissuedecellularization[J]. J Biomech Eng,2010,132(9):91003.
[118] Sano M B, Neal R N, Garcia P A, et al. Towards the creation of decellularized organconstructs using irreversible electroporation and active mechanical perfusion[J].Biomed Eng Online,2010,9:83.
[119] Henderson P W, Nagineni V V, Harper A, et al. Development of an acellularbioengineered matrix with a dominant vascular pedicle[J]. J Surg Res,2010,164(1):1～5.
[120] Wainwright J M, Czajka C A, Patel U B, et al. Preparation of cardiac extracellularmatrix from an intact porcine heart[J]. Tissue Eng Part C Methods,2010,16(3):525～532.
[121] Price A P, England K A, Matson A M, et al. Development of a decellularized lungbioreactor system for bioengineering the lung: the matrix reloaded[J]. Tissue EngPart A,2010,16(8):2581～2591.
[122] Cortiella J, Niles J, Cantu A, et al. Influence of acellular natural lung matrix onmurine embryonic stem cell differentiation and tissue formation[J]. Tissue Eng PartA,2010,16(8):2565～2580.
[123] Shupe T, Williams M, Brown A, et al. Method for the decellularization of intact ratliver[J]. Organogenesis,2010,6(2):134～136.
[124] Ross E A, Williams M J, Hamazaki T, et al. Embryonic stem cells proliferate anddifferentiate when seeded into kidney scaffolds[J]. J Am Soc Nephrol,2009,20(11):2338～2347.
[125] Marolt D, Knezevic M, Novakovic G V. Bone tissue engineering with human stemcells[J]. Stem Cell Res Ther,2010,1(2):10.
[126] Kofidis T, de Bruin J L, Hoyt G, et al. Myocardial restoration with embryonic stemcell bioartificial tissue transplantation[J]. J Heart Lung Transplant,2005,24(6):737～744.
[127] Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells fromadult human fibroblasts by defined factors[J]. Cell,2007,131(5):861～872.
[128] Zhang Y, Shen W, Sun B, et al. Plasticity of marrow mesenchymal stem cells fromhuman first-trimester fetus: from single-cell clone to neuronal differentiation[J]. CellReprogram,2011,13(1):57～64.
[129] Decembrini S, Cananzi M, Gualdoni S, et al. Comparative analysis of the retinalpotential of embryonic stem cells and amniotic fluid-derived stem cells[J]. StemCells Dev,2011,20(5):851～863.
[130] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouseembryonic and adult fibroblast cultures by defined factors[J]. Cell,2006,126(4):663～676.
[131] Prasad V K, Kurtzberg J. Transplant outcomes in mucopolysaccharidoses[J]. SeminHematol,2010,47(1):59～69.
[132] Shen Q, Goderie S K, Jin L, et al. Endothelial cells stimulate self-renewal andexpand neurogenesis of neural stem cells[J]. Science,2004,304(5675):1338～1340.
[133] Menon N G, Rodriguez E D, Byrnes C K, et al. Revascularization of humanacellular dermis in full-thickness abdominal wall reconstruction in the rabbitmodel[J]. Ann Plast Surg,2003,50(5):523～527.
[134] Deutsch M, Meinhart J, Vesely M, et al. In vitro endothelialization of expandedpolytetrafluoroethylene grafts: a clinical case report after41months ofimplantation[J]. J Vasc Surg,1997,25(4):757～763.
[135] Lee C Y, Mangino M J. Preservation methods for kidney and liver[J].Organogenesis,2009,5(3):105～112.
[136] Niklason L E, Gao J, Abbott W M, et al. Functional arteries grown in vitro[J].Science,1999,284(5413):489～493.
[137] Park J, Li Y, Berthiaume F, et al. Radial flow hepatocyte bioreactor using stackedmicrofabricated grooved substrates[J]. Biotechnol Bioeng,2008,99(2):455～467.
[138] Ratcliffe A, Niklason L E. Bioreactors and bioprocessing for tissue engineering[J].Ann N Y Acad Sci,2002,961:210～215.
[139] Nahmias Y, Kramvis Y, Barbe L, et al. A novel formulation of oxygen-carryingmatrix enhances liver-specific function of cultured hepatocytes[J]. FASEB J,2006,20(14):2531～2533.
[140] Lovett M, Lee K, Edwards A, et al. Vascularization strategies for tissueengineering[J]. Tissue Eng Part B Rev,2009,15(3):353～370.
[141] Sewnaik A, Hoorweg J J, Knegt P P, et al. Treatment of hypopharyngeal carcinoma:analysis of nationwide study in the Netherlands over a10-year period[J]. ClinOtolaryngol,2005,30(1):52～57.
[142] Keereweer S, de Wilt J H, Sewnaik A, et al. Early and long-term morbidity aftertotal laryngopharyngectomy[J]. Eur Arch Otorhinolaryngol,2010,267(9):1437～1444.
[143] Ramirez M J, Ferriol E E, Domenech F G, et al. Psychosocial adjustment in patientssurgically treated for laryngeal cancer[J]. Otolaryngol Head Neck Surg,2003,129(1):92～97.
[144] Baiguera S, Gonfiotti A, Jaus M, et al. Development of bioengineered humanlarynx[J]. Biomaterials,2011,32(19):4433～4442.
[145] Haykal S, Soleas J P, Salna M, et al. Evaluation of the structural integrity andextracellular matrix components of tracheal allografts following cyclicaldecellularization techniques: comparison of three protocols[J]. Tissue Eng Part CMethods,2012,18(8):614～623.
[146] Bhosale A M, Richardson J B. Articular cartilage: structure, injuries and review ofmanagement[J]. Br Med Bull,2008,87:77～95.
[147] Liu Y, Nakamura T, Yamamoto Y, et al. Immunosuppressant-freeallotransplantation of the trachea: the antigenicity of tracheal grafts can be reducedby removing the epithelium and mixed glands from the graft by detergenttreatment[J]. J Thorac Cardiovasc Surg,2000,120(1):108～114.
[148] Huey D J, Sanchez-Adams J, Willard V P, et al. Immunogenicity of bovine andleporine articular chondrocytes and meniscus cells[J]. Tissue Eng Part A,2012,18(5-6):568～575.
[149] Bujia J, Wilmes E, Hammer C, et al. Class II antigenicity of human cartilage:relevance to the use of homologous cartilage graft for reconstructive surgery[J]. AnnPlast Surg,1991,26(6):541～543.
[150] Langer F, Gross A E. Immunogenicity of allograft articular cartilage[J]. J Bone JointSurg Am,1974,56(2):297～304.
[151]马瑞娜,崔鹏程.喉移植免疫学研究进展[J].听力学及言语疾病杂志,2010,18(3):304～306.
[152] Rees L E, Ayoub O, Haverson K, et al. Differential major histocompatibilitycomplex class II locus expression on human laryngeal epithelium[J]. Clin ExpImmunol,2003,134(3):497～502.
[153] Wang E C, Damrose E J, Mendelsohn A H, et al. Distribution of class I and IIhuman leukocyte antigens in the larynx[J]. Otolaryngol Head Neck Surg,2006,134(2):280～287.
[154] Pittenger M F, Mackay A M, Beck S C, et al. Multilineage potential of adult humanmesenchymal stem cells[J]. Science,1999,284(5411):143～147.
[155] Wilson A S, Hobbs B G, Shen W Y, et al. Argon laser photocoagulation-inducedmodification of gene expression in the retina[J]. Invest Ophthalmol Vis Sci,2003,44(4):1426～1434.
[156] Otto A, Collins-Hooper H, Patel K. The origin, molecular regulation and therapeuticpotential of myogenic stem cell populations[J]. J Anat,2009,215(5):477～497.
[157] Ferrari G, Cusella-De A G, Coletta M, et al. Muscle regeneration by bonemarrow-derived myogenic progenitors[J]. Science,1998,279(5356):1528～1530.
[158] Noiseux N, Gnecchi M, Lopez-Ilasaca M, et al. Mesenchymal stem cellsoverexpressing Akt dramatically repair infarcted myocardium and improve cardiacfunction despite infrequent cellular fusion or differentiation[J]. Mol Ther,2006,14(6):840～850.
[159] Baraniak P R, McDevitt T C. Stem cell paracrine actions and tissue regeneration[J].Regen Med,2010,5(1):121～143.
[160]李秋果.组织工程支架材料研究进展及发展前景[J].长江大学学报自然科学版：农学卷,2012,9(2):21～25.
[161]侯楠,崔鹏程,罗家胜,等.灌注法与浸泡法构建去细胞全喉支架的对比研究[J].中国修复重建外科杂志,2009,(5):623～626.
[162]许莉,崔鹏程,陈志峰,等.犬去细胞喉支架的生物力学研究[J].中华耳鼻咽喉头颈外科杂志,2011,46(4):331～335.
[163]马瑞娜,崔鹏程,侯楠,等.去细胞全喉软骨支架的免疫学研究[J].中华耳鼻咽喉头颈外科杂志,2010,45(3):233～237.