羊颈动脉脱细胞血管基质的制备及其生物相容性评价

目的研究脱细胞血管基质快速高效的制备方法并对其进行生物相容性评价,为组织工程血管的研究寻找合适的支架材料。方法取20根长50mm新鲜山羊颈动脉经-80℃冷冻、37℃水浴复温,反复冻融3次,在506MPa、4℃条件下超高压处理20min,0.125%SDS中37℃振摇(100r/min)12h,彻底去除细胞后,行HE染色、Masson染色、扫描电镜观察及生物力学测定研究其组织结构的变化;Hoechst33258荧光染色和细胞DNA残留量分析评价脱细胞效果;通过接触细胞毒性实验、MTT活性测试及皮下埋植实验对制备的脱细胞血管基质进行生物相容性分析。皮下埋植实验取清洁级SD大鼠10只,体重200～230g,分为2组(n=5),实验组于大鼠背部皮下埋植结合物理化学方法处理的脱细胞血管基质,对照组埋植单纯物理方法处理的脱细胞血管基质,分别于术后1、2、3、4、8周取出行HE染色观察。结果 HE染色及Masson染色显示脱细胞血管基质无细胞成分残留,保持较完整的胶原成分;扫描电镜观察血管表面以胶原为主,适合细胞黏附;Hoechst33258荧光染色脱细胞血管基质材料未见明显阳性核染色;苯酚-氯仿提取脱细胞血管基质残留DNA含量,电泳检测分析显示其中DNA含量较正常血管显著降低;生物力学检测示脱细胞血管基质最大荷载时的应力及应变与正常血管比较差异无统计学意义,保持了正常血管的力学特征;接触细胞毒性实验与MTT法测定示脱细胞血管基质与细胞有良好的黏附性,细胞毒性为0～1级;实验组皮下埋植术后8周材料周围基本未见炎性细胞,对照组仍有明显淋巴细胞浸润。结论经反复冻融、超高压及小剂量SDS处理的脱细胞血管基质是一种构建组织工程血管的理想支架材料。     

十二烷基肌氨酸钠法制备带瓣管道支架及其生物学性能分析

目的尝试用十二烷基肌氨酸钠作为脱细胞试剂，制备脱细胞组织工程支架材料，并分析其生物学性能。方法用0．25％十二烷基肌氨酸钠溶液及核酸酶对猪的带瓣膜管道进行脱细胞处理，并对其进行DNA、可溶性蛋白含量测定，HE、Movat染色和电镜观察及生物力学测试；大鼠皮下包埋实验分析生物相容性。结果经脱细胞处理后，带瓣膜管道的瓣叶和管壁中DNA含量仅为正常的1．35％和2．81％，可溶性蛋白含量仅为正常的8．46％和12．65％，细胞成分几乎完全去除。HE和Movat染色及电镜观察显示组织结构完整、细胞结构完全消失，胶原、弹性纤维、蛋白聚糖等主要基质成分充分保留；瓣膜生物力学性能与正常瓣膜差异无统计学意义；支架生物相容性良好，并具有低免疫原性和可降解性。结论十二烷基肌氨酸钠是一种理想的新型脱细胞试剂，以此可以建立一套低毒、高效、对组织结构无损伤的脱细胞方法，并制备出生物学性能优异的脱细胞支架材料。     

脱细胞脊髓天然支架的制备及形态学观察

[目的]采用化学脱细胞方法去除细胞和髓鞘成分制作细胞外基质支架,为桥接脊髓损伤缺损提供理想的天然神经支架。[方法]取SD大鼠胸段脊髓约2cm,运用冻融 化学萃取(3%脱氧胆酸钠和1KU/mlDNaseI、RNaseA)的组织工程学方法处理大鼠脊髓组织,并对处理后的脊髓支架分别进行组织学检查,了解脱细胞情况及细胞外基质支架形态。[结果]经过脱细胞处理后,光镜下HE染色脊髓横断面呈网状结构,未见细胞成分存留,纵切面上呈互相交错的管状通路;未见轴突、髓鞘和细胞核。髓鞘染色可见髓鞘脱除彻底,未见髓鞘成分。[结论]本实验采用冻融 化学萃取的组织工程学方法可制备出理想的天然脊髓支架,该支架与脊髓三维组织结构具有高度相似性,有望作为脊髓损伤后桥接物和神经组织工程种子细胞的支架,本实验结果显示3%脱氧胆酸钠和1KU/mlDNa-seI、RNaseA进行脱细胞处理两次是较为合适的,能较为彻底去除细胞及髓鞘成分并较为完整地保留细胞外基质的三维结构。     

脱细胞异体真皮基质皮下移植后胶原的动态变化

目的　探讨脱细胞异体真皮基质 (ADM)移植后胶原的变化情况。方法　将异体ADM移植于SD鼠皮下 ,测定移植后ADM中胶原的含量及Ⅰ、Ⅲ型胶原的比例。结果　异体ADM移植后胶原含量及Ⅰ、Ⅲ型胶原比例无明显变化。结论　异体ADM是一种良好的软组织填充材料。     

冻干脱钙骨表面纳米结构对细胞行为的影响

目的：探讨表面有纳米结构的冻干脱钙骨基质(nFDBM)的制备方法及该结构对成骨细胞生物学行为的影响。方法：取新鲜狗趾皮质骨按改良Urist法制备冻干脱钙骨基质(FDBM)，经Nd：YAG激光表面处理后，用原子力显微镜、扫描电镜观察其表面形态结构学变化。nFDBM、FDBM分别与狗自体骨髓基质细胞诱导分化来的成骨细胞体外构建材料——细胞复合物并植人该狗脊柱两侧深筋膜袋内，于植人前及术后第4、8周取材行扫描电镜及组织学观察。结果：FDBM经Nd：YAG激光处理后表面形成了规则的纳米级沟槽状三维结构。体外构建材料——细胞复合物后扫描电镜观察nFDBM表面细胞黏附密度及分泌基质量均高于FDBM。术后HE染色、扫描电镜观察见左侧植人物基本纤维化，表面未见明显成骨细胞及基质；右侧植人物原纳米沟槽一侧形成薄层骨样组织，表面为成骨细胞分泌的大量基质所覆盖。结论：Nd：YAG激光可在FDBM表面形成纳米级沟槽状三维结构；该结构有利于成骨细胞的黏附、生长和基质分泌。     

脱细胞猪主动脉瓣膜与可降解聚合材料共同构建复合组织工程瓣膜

目的对新型复合组织工程瓣膜进行体外生物力学和动物体内移植试验，为临床应用过渡提供依据。方法以脱细胞猪主动脉瓣作为支架，用可降解聚合材料3-羟基丁酸与3-羟基己酸共聚酯（3一hydroxybutyrate—co-3-hydroxyhexanoate，PHBHHx）涂层，构建新型复合组织工程瓣膜。（1）复合组织工程瓣膜、新鲜猪主动脉瓣和脱细胞猪主动脉瓣各12枚，用单轴生物拉伸机进行体外生物力学测试；（2）小尾寒羊10只，其中5只在全身麻醉非体外循环下接受复合组织工程瓣膜，移植到羊的肺动脉瓣位；其余接受脱细胞猪主动脉瓣作为对照。术后18周处死动物，取出移植瓣膜，进行组织学、免疫荧光染色、扫描电子显微镜检查和钙含量测定。结果复合组织工程瓣膜保持了自然瓣形态，抗拉强度显著提高（P〈0．05）；瓣膜柔软，表面光滑无血栓；免疫荧光染色检测，瓣膜新生内膜中内皮细胞呈CD31阳性反应，沿瓣表面连续排列，间质细胞呈现单克隆鼠抗人平滑肌actin（sMA）阳性反应；复合组织工程瓣膜钙含量明显低于脱细胞猪主动脉瓣（P〈0．05）。结论复合组织工程瓣膜具有自然瓣膜的三维形态结构，良好的生物力学特性、生物相容性和细胞引导性，初步具备组织工程瓣膜雏形。     

一种新的脱细胞阴茎海绵体基质的制备方法

目的探索一种新的脱细胞阴茎海绵体基质的制备方法。方法取健康壮年兔完整阴茎海绵体组织，以Triton-X100与NH3·H2O（氨水）联合提取法进行脱细胞处理。标本作HE染色，组织学观察分析脱细胞效果。结果脱细胞处理25天后，成功获得脱细胞海绵体基质。所得基质外观良好。HE染色观察无细胞存在，弹力纤维排列规整，间隙较大，结构无破坏。结论利用Triton-X100与NH3·H2O联合提取法可成功制备完整无细胞阴茎海绵体基质。     

小鼠睾丸消化细胞异体移植重构生精小管的研究

目的:采用免疫缺陷小鼠作为受体,通过对小鼠睾丸消化细胞异位移植后不同时期移植物的研究,观察生精小管重构、生精细胞归巢及精子发生情况。方法:取新生ICR小鼠的睾丸消化成单细胞悬液,将其与Matrigel基质胶混匀后移植于雄性裸鼠背部皮下,术后裸鼠行去势。移植后分别于4、6、8、10周处死5只裸鼠,计算移植成功率,取移植物测量直径,并进行HE染色和免疫组化检测,观察生精小管的重构、生精细胞归巢及精子发生情况。结果:20只受体鼠接受睾丸消化细胞移植后全部存活。睾丸消化细胞移植后10周内可见明显隆起的包块,包块直径由第4周的(3.91±0.71)mm增加到(6.69±0.50)mm,移植物表面有血管生成。对移植物石蜡切片进行HE染色可见生精小管样结构,部分生精小管管腔内可见由精原细胞发育至精子细胞的各级生殖细胞,未见明显精子产生。对8周移植物进行免疫组化观察,可见生殖细胞标志物Mvh、支持细胞标志物Gata4和间质细胞标志物P450Scc表达。结论:新生小鼠睾丸消化细胞移植于裸鼠背部皮下后可重构生精小管,为研究睾丸组织工程及睾丸发育和精子发生过程中睾丸各组成细胞之间的相互作用提供了理想的研究模型。     

猪长骨脱细胞骨细胞外基质的制备及生物力学、组织学性质的初步检测

目的　寻找适合骨移植的骨组织替代物—骨脱细胞细胞外基质 (ABECM )。方法　将猪股骨用组织工程学的方法进行适当的脱细胞处理 ,制成ABECM ,再进行HE染色和扫描电镜、透射电镜观察 ,及纵向压缩的生物力学性质的检测。结果　ABECM未见细胞成分 ,细胞外基质基本结构未被破坏 ;ABECM在生物力学性质上与新鲜股骨无明显差异。结论　ABECM是一种新型的理想的骨替代物 ,有广泛的应用前景     

脱细胞尿道及其海绵体基质制备的实验研究

目的 探索脱细胞尿道及其海绵体基质的制备方法。方法取健康壮年兔完整尿道及其海绵体组织，以Triton-X100与NH3H2O联合提取法进行脱细胞处理。标本做HE染色，组织学观察分析脱细胞效果。结果脱细胞处理11天后，成功获得脱细胞及其海绵体基质，所得基质外观良好。HE染色观察无细胞存在。弹力纤维排列规整，间隙较大，结构无破坏。结论利用Triton-X100与NH3H2O联合提取法可成功制备完整无细胞尿道及其海绵体基质，为尿道再造修复提供崭新思路。     

异种脱细胞真皮与异体巩膜作为HA义眼台包裹材料的实验研究

目的 观察异种脱细胞真皮（acellular xenogenic dermal matrix,X-ADM）和异体巩膜作为羟基磷灰石（hydroxy apatite,HA）义眼台包裹材料,用于实验兔眼的临床表现及病理组织学变化观察.方法 24只纯种新西兰兔,行一只眼的眼球摘除术后,随机平均分为实验组和对照组.于肌锥内分别置入由异种脱细胞真皮及异体巩膜包裹的HA义眼台.术后观察眼部表现,于1、2、4、6、8和12周,连同异种脱细胞真皮或异体巩膜取出义眼台,光镜下观察包裹材料与义眼台的组织病理学改变,包括炎症反应及血管化情况.取4、8和12周标本做透射电镜观察上述组织的超微结构改变.结果 与异体巩膜组相比,异种脱细胞真皮组在同期的成纤维细胞及新生血管的生长更活跃,而且长入较早,新生胶原形成丰富,几乎没有炎症细胞的浸润以及发生排斥反应.结论 异种脱细胞真皮血管化快,免疫原性低,是一种良好的巩膜替代物.     

Time-related histopathologic analyses of immunologically untreated porcine valved conduits implanted in a porcine-to-goat model

This study was performed to evaluate the clinical feasibility of use of immunologically nontreated xenogenic valves, using a porcine-to-goat pulmonary valved conduit implantation model. Porcine pulmonary valve conduits were prepared with no specific immunological treatment and implanted in the right ventricular outflow tract of goats under cardiopulmonary bypass. The goats were assigned at predetermined intervals (1 day, 1 week, and 3, 6, and 12 months) as two animals for each interval. Echocardiographic examinations of the valves were performed before sacrifice. Upon retrieving the xenograft specimens, they were inspected visually and microscopically. Ten of the 12 animals survived the predetermined observation periods. Variable degrees of pulmonary regurgitation were the main findings on echocardiographic evaluations. On gross examination of the explanted specimens, all leaflets, except in one animal that prematurely died, were fairly well preserved. They were slightly shortened but free of thrombosis or vegetation. Aneurysmal dilatations of the anterior wall of the implanted pulmonary artery were observed in one of 12-month-survival animals and in another one of 3-month-survival animals. Microscopically, the three components of implanted xenografts (the pulmonary artery, valve, and infundibulum) were shown to be gradually replaced with host cells in time, while maintaining structural integrity. The nuclei of the donor tissue disappeared through pyknosis and karyolysis. In conclusion, immnunologically untreated xenogenic pulmonary valved conduits can be an alternative potential as valve substitutes with distinctive advantages of providing self-healing potential, despite a few problems observed in the current study such as occurrences of pulmonary regurgitation and sporadic cases of aortic aneurysm.     

脱细胞异种(猪)真皮基质作为填充材料的初步研究与应用

目的：为临床修复体表凹陷畸形寻求良好的填充材料。方法：用自制脱细胞异种（猪）真皮基质，植入兔皮下，定时进行测量，并进行光镜及电镜观察。临床应用2例。结果：脱细胞异种（猪）真皮基质植入后观察1年，仅在4周内有极轻微的炎性反应，无排斥反应，2周左右有成纤维细胞及毛细胞血管长入，吸收率低于对照组。2例临床应用填充效果较满意，质地较软，无破溃、外露。结论：脱细胞异种（猪）真皮基质组织相容性好，吸收率低，质地软，是一种有良好前景的软组织填充材料。     

In vivo repopulation of xenogeneic and allogeneic acellular valve matrix conduits in the pulmonary circulation

BACKGROUND: Approaches to in vivo repopulation of acellularized valve matrix constructs have been described recently. However, early calcification of acellularized matrices repopulated in vivo remains a major obstacle. We hypothesised that the matrix composition has a significant influence on the onset of early calcification. Therefore, we evaluated the calcification of acellularized allogenic ovine (AVMC) and xenogenic porcine (XVMC) valve matrix conduits in the pulmonary circulation in a sheep model. METHODS: Porcine (n = 3) and sheep (n = 3) pulmonary valve conduits were acellularized by trypsin/EDTA digestion and then implanted into healthy sheep in pulmonary valve position using extracorporeal bypass support. Transthoracic echocardiography (TTE) was performed at 12 and 24 weeks after the implantation. The animals were sacrificed at week 24 or earlier when severe calcification of the valve conduit became evident by TTE. The valves were examined histologically and biochemically. RESULTS: All AVMC revealed severe calcification after 12 weeks with focal endothelial cell clustering and no interstitial valve tissue reconstitution. In contrast, after 24 weeks XVMC indicated mild calcification on histologic examination (von Kossa staining) with histologic reconstitution of valve tissue and confluent endothelial surface coverage. Furthermore, immunohistologic analysis revealed reconstitution of surface endothelial cell monolayer (von Willebrand factor), and interstitial myofibroblasts (Vimentin/Desmin). CONCLUSIONS: Porcine acellularized XVMC are resistant to early calcification during in vivo reseeding. Furthermore, XVMC are repopulated in vivo with valve-specific cell types within 24 weeks resembling native valve tissue.     

Expression and cellular localization of interleukin 8 mRNA and protein in the area of xenogenic bone implant

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Living,autologous pulmonary artery conduits tissue engineered from human umbilical cord cells

BACKGROUND: Tissue engineering represents a promising approach to in vitro creation of living, autologous replacements with the potential to grow, repair, and remodel. Particularly in a congenital operation, there is a substantial need for such implantation materials. We previously demonstrated fabrication of completely autologous, functional heart valves on the basis of peripheral vascular cells. Presently the feasibility of creating pulmonary artery conduits from human umbilical cord cells was investigated. METHODS: Human umbilical cord cells were harvested and expanded in culture. Pulmonary conduits fabricated from rapidly bioabsorbable polymers were seeded with human umbilical cord cells and grown in vitro in a pulse duplicator bioreactor. Morphologic characterization of the generated neo-tissues included histology, transmission, and scanning electron microscopy. Characterization of extracellular matrix was comprised of immunohistochemistry. Extracellular matrix protein content and cell proliferation were quantified by biochemical assays. Biomechanical testing was performed using stress-strain and burst-stress tests. RESULTS: Histology of the conduits revealed viable, layered tissue and extracellular matrix formation with glycosaminoglycans and collagens I and III. Cells stained positive for vimentin and alpha-smooth muscle actin. Scanning electron microscopy showed confluent, homogenous tissue surfaces. Transmission electron microscopy demonstrated elements typical of viable myofibroblasts, such as collagen, fibrils, and elastin. Extracellular matrix proteins were significantly lower compared with native tissue; the cell content was increased. The mechanical strength of the pulsed constructs was comparable with native tissue; the static controls were significantly weaker. CONCLUSIONS: In vitro fabrication of tissue-engineered human pulmonary conduits was feasible utilizing human umbilical cord cells and a biomimetic culture environment. Morphologic and mechanical features approximated human pulmonary artery. Human umbilical cord cells demonstrated excellent growth properties representing a new, readily available cell source for tissue engineering without necessitating the sacrifice of intact vascular donor structures.     

去细胞猪主动脉瓣移植于犬腹主动脉内构建组织工程瓣

目的 探讨在犬腹主动脉内构建组织工程心脏瓣膜的实验方法。方法 将预种犬血管间质细胞和内皮细胞的去细胞猪主动脉瓣叶(猪瓣),移植于6条犬的腹主动脉内,于术后4,6,8和10周对移植瓣叶进行形态、组织结构及免疫组化染色观察。结果 (1)移植术后4周时瓣叶周边有多层细胞长入,新细胞外基质形成,原支架组织部分吸收。(2)10周末猪瓣组织完全吸收,为宿主细胞及新合成的细胞外基质取代。基质中间质细胞主要为成纤维细胞和肌纤维母细胞。细胞外基质成分主要为Ⅰ、Ⅲ型胶原和少量弹力纤维,并含有中性和酸性黏多糖。(3)内皮细胞覆盖于瓣叶表面。结论 (1)移植于犬腹主动脉内的去细胞猪瓣于移植术后10周末基本构成组织工程瓣叶;(2)腹主动脉内异位移植是一种可供选择的实验研究方法。     

带蒂肌瓣包裹异种无机骨与自体红骨髓复合物的实验研究

目的 制备带血运并具有新骨组织形成的,来源于异种无机骨与自体红骨髓复合物的肌骨瓣,方法 6月龄新西兰大白兔12只,实验组,每只兔左前肢尺桡骨旁肌肉内植入异种无机骨与自体红骨髓复合物,对照组,右侧相同部位植入单纯异种无机骨,术后2、8、12周进行X线片,大体标本和组织学观察。结果 两组X线片显示植入物呈松质骨样密度,不随植入时间改变,大体观察见植入后2周植入物与肌肉完全融合,包裹有植入物的带蒂肌瓣的血管清晰可见,将此包裹有植入物的带蒂肌瓣游离30分钟后,植入物仍然与周围组织色泽相同,无缺血坏死改变,组织学观察植入后2、8、12周显示植入部位肌肉组织与植入物相连接,但实验组植入后2周,血管进入复合物中,无新骨形成,8周,异种无机骨边缘有较少的新骨形成征象;12周,新骨组织形成明显增多,成骨细胞位于骨的边缘,周围为未分化间充质细胞,血管和骨基质丰富,骨细胞包绕在骨陷窝中,偶尔可见骨髓腔,对照组直至12周,异种无机骨孔隙间仅有带血管的疏松结缔组织无新骨形成,结论 异种无机骨与自体红骨髓复合物植入肌肉内12周可预制成具有血运的、有新骨形成的肌骨瓣。     

Tissue Engineering für Herzklappen und Gefäße

Current prosthetic substitutes for heart valves and blood vessels have numerous limitations such as limited durability (biological valves), susceptibility to infection, the necessity of lifelong anticoagulation therapy (prosthetic valves), and reduced patency in small-caliber grafts, for example. Tissue engineering using either polymers or decellularized native allogeneic or xenogenic heart valve/vascular matrices may provide the techniques to develop the ideal heart valve or vascular graft. The matrix scaffold serves as a basis on which seeded cells can organise and develop into the valve or vascular tissue prior to or following implantation. The scaffold is either degraded or metabolised during the formation and organisation of the newly generated matrix, leading to vital living tissue. This paper summarises current research and first clinical developments in the tissue engineering of heart valves and vascular grafts.     

高频振荡通气及与肺表面活性物质联用对吸入性损伤家兔肺炎性反应的影响

目的 了解高频振荡通气(HFOV)及HFOV与肺表面活性物质(PS)联合应用对吸人性损伤家兔肺组织炎性反应的影响.方法 将新西兰大白兔24只制成重度蒸气吸入性损伤模型,按随机数字表法分为对照组、HFOV组和HFOV+PS组,每组8只,分别行定容通气、HFOV及HFOV+PS治疗.治疗3.5 h时取各组家兔肺组织标本,行病理学检查及肺损伤评分,检测髓过氧化物酶(MPO)及半胱氨酸天冬氨酸蛋白酶1(caspase-1)活性,测定TNF-α、IL-18、IL-10和IL-13含量及其mRNA的表达情况.结果 (1)各组吸入性损伤家兔肺组织出现不同程度病理学改变,以对照组最明显,HFOV+PS组最轻.对照组、HFOV组、HFOV+PS组肺损伤评分各为(3.71±0.43)、(2.87±0.26)、(2.08±0.28)分,组间两两比较,差异均有统计学意义(P<0.01).(2)HFOV组和HFOV+PS组家兔MPO、caspase-1活性明显低于对照组(P<0.01),并且HFOV+PS组明显低于HFOV组(P<0.05).(3)HFOV组和HFOV+PS组家兔TNF-α、IL-18含量及其mRNA表达量明显低于对照组(P<0.01),IL-10和IL-13含量及其mRNA表达量明显高于对照组(P<0.01);HFOV+PS组这几项指标较HFOV组变化更明显(P<0.05).结论 HFOV能减轻吸入性损伤家兔肺组织炎性反应和肺损伤,联合应用PS效果更佳.