同种异体组织工程化软骨组织形成的初步研究

目的：探讨以同种异体的软骨细胞作为种子细胞、以聚乳酸(PLA)作为支架，应用组织工程学方法在体内形成新生软骨组织的可能性，为组织工程软骨修复喉、气管软骨缺损提供实验依据。方法：将出生3～5d的新西兰大白兔乳兔四肢关节软骨组织酶解，分离出软骨细胞，体外扩增后，将其接种到PLA三维可吸收支架上；将软骨细胞-PLA复合物移植到成年新西兰大白兔的背部皮下，经过7周的体内培养，将组织标本行大体观察和组织学检查。结果：软骨细胞-PLA复合物在同种异体的动物体内有新生组织形成，经组织学染色，证实为软骨组织；随着植入时间的延长，软骨基质的分泌增加，炎性细胞的浸润逐渐减轻。结论：同种异体的软骨细胞-PLA复合物在有免疫力的动物体内能够形成新生软骨组织，这一方法为喉、气管软骨缺损的修复提供了一条新的途径。     

聚羟基乙酸负载软骨细胞修复同种异体甲状软骨缺损

目的 探讨聚羟基乙酸(polyglycolic acid，PGA)负载软骨细胞在有免疫力动物修复同种异体甲状软骨缺损的可行性。方法 酶消化法获取3天龄新西兰乳兔肋软骨和关节软骨细胞，采用组织工程技术制备软骨细胞-PGA复合物，体外共同培养1周后用于修复同种异体成年新西兰白兔甲状软骨缺损(实验组7只)。设单纯PGA材料修复组(对照A组4只)和单纯软骨细胞修复组(对照B组4只)作为对照实验。分别于术后不同时间取材，对甲状软骨缺损的修复效果进行大体和组织学评价。结果 体外培养阶段可见黏附于PGA纤维表面的细胞分泌出丰富的软骨基质，呈蜘蛛网状分布于PGA纤维之间。术后4周大体观察：实验组修复区呈淡黄色，与正常软骨界限分明；组织学检查：修复区内有软骨细胞生成和基质分泌，但与正常软骨间存在界面无细胞区，未见明显炎细胞浸润。8周：实验组修复区色乳白，与正常软骨仍有界限；镜下见修复区软骨细胞较成熟，软骨基质含量丰富。12周：实验组修复区呈瓷白色，界面区软骨细胞不明显，但修复区软骨细胞数量、形态和基质与正常软骨相似。各时间点对照组大体观察修复区均呈不同程度的凹陷，暗红色，部分软组织充填其中，质软；组织学及特殊染色检查未发现软骨样结构及其分泌的基质成分。新生软骨内未见血管生长。结论PGA负载软骨细胞能修复具有正常免疫功能同种异体兔甲状软骨缺损，但新生软骨与正常软骨间存在无细胞区界面，无明显免疫排斥。     

内衬上皮的管状组织工程软骨构建的实验研究

目的研究以聚丙交酯-乙交酯共聚物[poly（DL-lactide-co-glycolide）,PLGA]包埋甲壳胺无纺布为细胞支架,在有免疫力的动物体内构建内衬上皮的管状组织工程软骨的可行性,为喉、气管缺损的修复探索新方法。方法取1月龄兔耳廓软骨,收集体外培养第3～4代的软骨细胞,接种于经多聚赖氨酸处理的以PLGA包埋甲壳胺无纺布的片状细胞支架材料上,体外培养7～10d后包裹于兔腹部缝制的皮管上并深埋于体内,分别于6、12、18周取材行大体和组织学观察以及HE染色、甲苯胺蓝染色和Masson＇s三色染色。结果同种异体软骨细胞/PLGA包埋甲壳胺无纺布的细胞支架复合物体外培养1周时有基质产生;植入体内6周后,复合物中有基质分泌,软骨细胞不成熟;12周时,软骨细胞接近成熟并形成凹陷,内衬上皮的管状组织工程软骨形成;18周时,新生软骨基本接近正常软骨。结论以PLGA包埋甲壳胺无纺布为细胞支架,同种异体软骨细胞与细胞支架所形成的复合物在有免疫力的动物体内可构建出内衬上皮的管状组织工程软骨。     

耳鼻咽喉科领域的软骨组织工程

耳、鼻、喉和气管的软骨组织工程研究已有较大进展，已分别在裸鼠和具有免疫力动物体内形成了与耳鼻咽喉有关的组织工程化软骨组织，并有初步应用的实验研究报道。随着胚胎干细胞、骨髓基质干细胞、同种异体软骨细胞和智能生物材料的研发和应用，耳鼻咽喉科软骨组织工程有望获得突破性进展。本文综述了该领域的软骨组织工程研究现状和发展趋势。     

同种异体软骨修复甲状软骨缺损的实验研究

目的　观察用不同类型的同种异体软骨修复喉软骨缺损的效果。方法　取新西兰白兔16只 ,平均分成两组。在每只兔的双侧甲状软骨切除 6mm× 3mm× 1mm全层软骨。一组动物将RPMI 16 4 0液和甲醛保存 30d的同种异体甲状软骨 (大小为 5mm× 2mm× 1mm)分别植入甲状软骨两侧缺损处 ,另一组动物于甲状软骨两侧缺损处分别植入新鲜的自体和异体软骨。分别于术后 7、30、180及 36 0d时处死取材 ,用大体观察、HE染色及免疫组织化学方法观察移植修复效果。结果经连续 1年观察 ,RPMI 16 4 0液保存的及新鲜的同种异体软骨与自体软骨移植排斥反应轻 ,均能较好修复甲状软骨缺损 ,而甲醛保存软骨早期有明显炎性细胞浸润 ,移植 180d至 36 0d时软骨基质明显吸收 ,软骨细胞退变 ,甲状软骨缺损区为纤维结缔组织修复。结论　RPMI 16 4 0液保存的及新鲜的同种异体软骨都具有活性 ,和自体软骨一样是软骨缺损修复的良好材料 ,可应用于临床     

同种异体软骨细胞修复耳廓缺损的动物实验研究

目的探讨兔同种异体软骨细胞和自行制备生物材料高孔隙率聚乳酸(poly—DL-lactide,PDLLA)支架复合物修复兔耳廓软骨缺损的可行性。方法 18只大白兔分为软骨细胞/PDLLA复合物移植组、单纯PDLLA对照组和空白对照组,将体外培养的兔同种异体软骨细胞和自行制备的PDLLA支架形成复合物,修复兔耳廓软骨缺损。分别于植入后6周、12周、18周取材,HE和甲苯胺蓝染色了解兔耳廓缺损修复情况。结果术后18周,软骨细胞/PDLLA复合物移植组软骨缺损区为软骨组织修复,新生软骨与正常软骨问连接好,单纯PDLLA对照组和空白对照组为纤维软骨或纤维组织修复。结论同种异体软骨细胞/自制PDLLA复合物移植可较好地修复兔耳廓软骨缺损。     

转化生长因子β1和同种异体软骨细胞及高孔隙率聚乳酸复合物修复兔耳廓软骨缺损

目的 观察加入转化生长因子-β1(transforming growth factor-beta1，TGF-β1)的同种异体软骨细胞／高孔隙率聚乳酸(poly-DL-lactide，PDLLA)支架复合物修复兔耳廓软骨缺损的效果。方法 18只大白兔随机分为同种异体软骨细胞／PDLLA复合物加TGF-β1组(复合物组)、同种异体软骨细胞／PDLLA复合物组(复合物对照组)和不用任何修复材料的空白对照组，每组6只。分别于4、12、18周取材，行HE和甲苯胺蓝染色，观察各组兔耳廓软骨缺损修复情况。结果 术后18周，TGF-β1复合物组修复软骨缺损的效果无论从大体标本观察或是病理组织学检查都比复合物对照组的修复效果好，空白对照组为纤维组织修复。结论 同种异体软骨细胞／PDLLA复合物移植可形成组织工程化软骨修复兔耳廓软骨缺损，TGF-β1可提高同种异体软骨细胞／PDLLA复合物移植修复兔耳廓软骨缺损的质量。     

同种异体工程化软骨的构建和修复甲状软骨缺损的实验研究

目的探讨同种异体预定形态组织工程化软骨在有免疫功能动物体内构建的可行性及其对甲状软骨缺损的修复能力.方法利用组织工程技术制备同种异体片状和"C”型半管状工程化软骨;将4周形成的片状工程化软骨用于修复12只新西兰大白兔甲状软骨大片缺损;一定时间取材,分别对预定形态工程化软骨的构建情况及其修复效果进行大体和组织学评价.结果①构建4周形成的预定形态工程化软骨呈乳白色,有弹性和支撑力,8周时软骨呈瓷白色,镜下观察显示软骨组织特征;②甲状软骨缺损修复术后4、8、12周观察,修复区愈合良好,组织学检查修复区与正常软骨间的界面区可见软骨细胞生长及软骨基质生成,无免疫排斥迹象.结论在有免疫功能的动物体内可形成同种异体预定形态组织工程化软骨,获取的工程化软骨对甲状软骨缺损有良好的修复效果,无明显免疫排斥反应.     

聚羟基乙酸负载软骨细胞修复同种异体甲状软骨缺损

目的　探讨聚羟基乙酸 ( polyglycolicacid ,PGA)负载软骨细胞在有免疫力动物修复同种异体甲状软骨缺损的可行性。方法　酶消化法获取 3天龄新西兰乳兔肋软骨和关节软骨细胞 ,采用组织工程技术制备软骨细胞 PGA复合物 ,体外共同培养 1周后用于修复同种异体成年新西兰白兔甲状软骨缺损 (实验组 7只 )。设单纯PGA材料修复组 (对照A组 4只 )和单纯软骨细胞修复组 (对照B组 4只 )作为对照实验。分别于术后不同时间取材 ,对甲状软骨缺损的修复效果进行大体和组织学评价。结果　体外培养阶段可见黏附于PGA纤维表面的细胞分泌出丰富的软骨基质 ,呈蜘蛛网状分布于PGA纤维之间。术后 4周大体观察 :实验组修复区呈淡黄色 ,与正常软骨界限分明 ;组织学检查 :修复区内有软骨细胞生成和基质分泌 ,但与正常软骨间存在界面无细胞区 ,未见明显炎细胞浸润。 8周 :实验组修复区色乳白 ,与正常软骨仍有界限 ;镜下见修复区软骨细胞较成熟 ,软骨基质含量丰富。1 2周 :实验组修复区呈瓷白色 ,界面区软骨细胞不明显 ,但修复区软骨细胞数量、形态和基质与正常软骨相似。各时间点对照组大体观察修复区均呈不同程度的凹陷 ,暗红色 ,部分软组织充填其中 ,质软 ;组织学及特殊染色检查未发现软骨样结构及其分泌的基质成分。新生软骨     

SIS with tissue-cultured allogenic cartilages patch tracheoplasty in a rabbit model for tracheal defect

Conclusions: In the rabbit model, small intestinal submucosa (SIS) compounded with tissue-cultured allogenic cartilages appeared to be an efficacious method for the patch repair of partial circumferential tracheal defects instead of autologous grafts. SIS appears to be a safe and promising means of facilitating neovascularization and tissue regeneration. The long-term use of SIS and tissue-cultured allogenic cartilages warrants further investigation. Background: Tracheal defect reparation remains a challenging surgical problem that can require reconstruction using autologous grafts or artificial stents. This study was performed to evaluate the efficacy of SIS, a biocompatible, acellular matrix, compounded with different tissue-cultured allogenic cartilages, in the repair of a critical-size tracheal defect. Materials and methods: A full-thickness defect (4×8 mm) was created in tracheal rings four to six in adult rabbits. A piece of 8-ply SIS sandwiched in thyroid cartilage, auricular cartilage, or without cartilage, respectively (designated experiment 1, 2, or 3, respectively), was sutured to the edges of the defect with interrupted 4-0 polypropylene sutures. In control animals, the defect was closed with lamina praetrachealis. All animals were followed until signs of dyspnea became apparent or for 4 or 12 weeks. After follow-up and euthanasia, the trachea was harvested and prepared for histologic evaluation using conventional techniques. Results: All animals tolerated the procedure well but two animals in group 1 (n=5), three in group 2 (n=5), and one in group 3 (n=5) had stridor after operation and expired within <1 month with different degrees of obstruction. The other animals in these groups and the control animals (n =3) all survived >1 month. Histologically, neovascularization of the patch was noted with moderate inflammation. The surface of the SIS patch was covered with a lining of ciliated epithelial cells. The tissue-cultured allogenic cartilages degraded to some extent.     

耳廓软骨天然生物支架材料的制备及初步研究

目的探讨制备兔耳廓天然软骨细胞外基质材料的新方法,制备适用于组织工程应用的天然生物支架材料.方法①采用多步骤去污剂-酶处理技术抽提兔耳廓软骨细胞制备天然软骨细胞外基质(Nature Extracellular Matrix,NECM),对NECM行HE染色、澳新蓝染色、维多利亚蓝染色、VanGieson染色并进行组织学及扫描电镜观察;②以灰家兔NECM为支架材料和新西兰大白兔耳廓软骨细胞为种子细胞进行体外培养,倒置显微镜下观察其亲水性和对细胞的吸附力,并通过四氮唑盐比色法(MTT Assay)观察NECM对软骨细胞的增殖作用.结果通过HE染色、蛋白多糖、胶原及弹力纤维特染、扫描电镜观察分析证实经过多步骤去污剂-酶法抽提软骨细胞制备的天然生物支架材料里,不含细胞及细胞器成分,仅含有不溶解的胶原、弹性蛋白和蛋白多糖,亦即NECM;体外培养实验发现,兔耳廓软骨细胞易进入脱细胞异体软骨基质结构裸露的空穴,且生长旺盛,组织学检查证实兔耳廓软骨NECM上裸露的骨陷窝已部分被软骨细胞占据.MTT Assay检测证实NECM对软骨细胞增殖有促进作用.结论本实验制备的NECM脱细胞彻底、细胞外基质成分保留较完整,并具有促进细胞粘附、增殖和分化生长的作用,是一种较为理想的天然生物支架材料.     

吸人性烧伤气管切开的时机选择与指征

目的:探讨大面积烧伤合并有吸入性损伤时气管切开的时机与指征。方法:对180例大面积烧伤(其中石油天然气烧伤144例,轻质油烧伤26例,热蒸汽烧伤10例),合并有呼吸道吸入性损伤的病人,在烧伤后进行气管切开的依据、必要性、时机、指征及手术要点进行总结。结果:180例大面积烧伤合并有呼吸道吸入性损伤病人,烧伤后1-24小时进行气管开术124例,其中轻度85例(气管切开后死亡4例),中度22例(气管切开后死亡9例),重度17例(气管切开后死亡11例),超过24小时后进行气管切开术51例,其中轻度12例(气管切开后死亡8例),中度34例(气管切开后死亡15例),重度5例(气管切开后死亡5例)。有5例重度吸入性损伤未及气管切开即死亡。气管切开时颈部有环状或半环状焦痂21例,伴头面部严重烧伤90例,口呈鱼嘴状者32例,口鼻内大量血清性渗出者37例。结论:吸入性损伤时喉梗阻危象的发生是引起烧伤病人死亡的重要原因,强调大面积烧伤伴有呼吸道吸入性损伤时,应在烧伤后早期及时进行气管切开术,可以降低病人喉梗阻危象的发生率和死亡率。     

自制NECM修复兔耳廓软骨缺损的试验研究

目的本研究的目的在于评价自制同种异体天然细胞外基质(Nature Extracellular Matrix,NECM)在修复兔耳廓软骨缺损中的价值,从而在体外研究的基础上进一步证实我们采用的去污剂-酶四步法制作的NECM在组织工程学中的可行性.方法选用10只新西兰大白兔,于其双耳廓制造1.0×.0cm大小的软骨缺损后,分别作以下研究:①灰兔NECM修复兔耳廓缺损.②灰兔NECM复合软骨膜修复兔耳廓缺损;③不植任何材料的空白对照.术后4、6、12、24周取材并行组织学检查.结果NECM植入后4、6周有轻重不一的炎症反应,第12周炎症反应消失,NECM周边无包裹现象.第12周时NECM复合软骨膜组有软骨膜细胞长入NECM,此时的软骨膜细胞多处于增殖期,24周时软骨膜细胞继续增殖分化细胞逐渐长入NECM深部,且部分软骨膜细胞发育成熟.单纯NECM修复组,第6周出现纤维结缔组织长入NECM,12、24周纤维结缔组织逐渐长入NECM深部,并逐渐取代NECM.结论①NECM可诱导软骨膜细胞增殖分化生长,修复软骨缺损:②NECM具有较好的组织亲和性和相容性;③软骨膜细胞可以作为NECM再生软骨的种子细胞的来源;④软骨膜可以阻止纤维结缔组织侵入修复区NECM.     

Surgical treatment of laryngeal tumors with subglottic extension and tracheal tumors with composite nasal septal cartilage graft: technique and outcome

OBJECTIVE: Composite nasal septal cartilage grafts (CNSCG) are effective grafting materials in laryngeal and tracheal reconstruction following tumor resection. METHODS: Between 1985 and 2005, we used CNSCG for the reconstruction of defects following resection of laryngeal tumors with subglottic extension (20 cases), subglottic mesenchymal tumors (2 cases), invasive thyroid carcinoma (4 cases), tracheal tumors (3 cases) and esophagus carcinoma with tracheal invasion (1 case) in total of 30 patients. RESULTS: The patients with subglottic tumors were decanulated within 5-7 days except one case. We achieved satisfactory voice and swallowing without any sign of recurrence. Overall complications consisted of subglottic stenosis in one case, and unilateral paralysis of recurrent laryngeal nerve in two cases. One patient with subglottic laryngeal carcinoma died due to neck and distant metastases 4 years after the operation. All patients are well with a mean follow-up period 9 years. Three patients with tracheal tumors underwent lateral resection and reconstructed with CNSCG. Satisfactory healing of the grafts was seen in all cases without local recurrence or complication with a mean follow-up period of 62 months. One of the patients had distant metastases 3 years after the operation. The patient with esophagus carcinoma and tracheal invasion was treated by total esophagectomy, gastric pull-up, tracheal resection and CNSCG reconstruction. He died at postoperative 5th day due to mediastinitis as a complication of gastric pull-up. CONCLUSION: Free composite cartilage graft is a reliable material in the reconstruction of defects after surgery of laryngeal tumors with subglottic extension, invasive thyroid and esophagus tumors and well-selected tracheal tumors.     

The effectiveness of hyperbaric oxygen treatment in tracheal reconstruction with auricular cartilage grafts (experimental study)

PURPOSE: Tracheal stenosis or neoplastic changes, as well as, traumatic, congenital, or iatrogenic causes may require extensive tracheal resections. Complications like vascularization insufficiency and structural support problems occur nearly in all cases when end-to-end anastomosis of trachea is not feasible. Hyperbaric oxygen (HBO) treatment is a well-known method for the management of grafts and flaps that have vascularization problems. In this study, the effect of hyperbaric oxygen treatment on wound healing after tracheal reconstruction with auricular cartilage graft (ACG) has been evaluated. METHODS: Thirty-two rabbits were divided into 2 groups: study group (n = 16) and control group (n = 16). The anterior halves of the six tracheal rings were resected, and the defects were repaired with autogenic auricular grafts. Hyperbaric 100% pure oxygen was administered to the study group at 2.4 atmospheres of absolute pressure 2 times a day for 1 week. The control group did not receive any therapy except proper control of the wound. RESULTS: It was observed that in the study group, inflammation, fibrosis, and necrosis were less, whereas epithelialization and maturation were early and neovascularization and neochondrification were more than the control group only at specific weeks. But all tracheas in both groups showed excellent healing without graft rejection and excessive granulation tissue formation. Furthermore, there was no statistically difference between the 2 groups. CONCLUSIONS: Auricular cartilage grafts is a valuable management method of tracheal defects, and hyperbaric oxygen treatment is a good supplementary method in healing period of cartilage autografts.     

GDNF转染雪旺氏细胞构建神经导管复合体

目的利用GDNF(胶质神经源性神经营养因子)基因转染的雪旺氏细胞和PLGA(聚乳酸/聚乙醇酸共聚物)管共同培养构建神经导管复合体。方法利用乳鼠臂丛神经和坐骨神经培养雪旺氏细胞,再把pcDNA3.1( )/GDNF质粒通过脂质体转入细胞内。利用PCR检测转染雪旺氏细胞的GDNF表达。将转染雪旺氏细胞扩增达到一定数量后,用微注射和共培养法,与PLGA管构建神经导管。通过光镜和扫描电镜检测雪旺氏细胞的生长情况。结果成功培养出雪旺氏细胞,并转入GDNF基因。转染的雪旺氏细胞可表达GDNF,PCR显示转染的雪旺氏细胞GDNF表达较正常细胞明显提高。转染的雪旺氏细胞可在PLGA管表面贴壁、生长。结论GDNF基因转染的雪旺氏细胞可与PLGA管共同构建神经导管复合体。