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(transforminggrowthfactor beta 1,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。     

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     

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     

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。     

同种异体组织工程软骨修复气管壁缺损的实验研究

目的　探讨以同种异体的软骨细胞作为种子细胞 ,可吸收材料PLA作为支架 ,应用组织工程学方法形成的新生软骨组织修复动物气管壁缺损的可行性。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 ,接种到PLA可吸收支架上 ,移植到成年新西兰大白兔的背部皮下 ,7周后形成新生软骨组织修复实验动物颈部气管前壁约 10mm× 10mm大小的缺损 ,术后观察 8周 ,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形成新生组织 ,经组织学染色 ,证实为软骨组织。新生组织工程软骨修复动物气管壁缺损后 8周 ,气管管腔通畅 ,无狭窄 ;组织学染色证实修复处软骨组织生长良好 ,无坏死 吸收。结论　同种异体幼年动物的软骨细胞 PLA复合物在有免疫力的动物体内形成的组织工程软骨可以作为气管壁缺损的修复材料。     

自体骨髓间质干细胞诱导的软骨细胞修复兔耳廓软骨缺损

目的探讨自体骨髓间质干细胞（Bone marrow mesenchymal stemcells,BMMSC）诱导的软骨细胞修复兔耳廓软骨缺损的可行性。方法取新西兰大白兔BMMSC体外培养扩增,以含转化生长因子（TGF-β1）、地塞米松和维生素C的培养液做诱导培养,实验组以诱导后的软骨细胞与聚丙交酯-乙交酯共聚物Poly（dl-lactideco-glycolide）（PLGA）包埋甲壳胺无纺布的新型支架形成复合物修复兔自体耳廓软骨缺损;对照组以PLGA/甲壳胺无纺布支架修复兔耳廓软骨缺损。分别于移植后6周、12周、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,实验组缺损区完全由软骨组织修复,缺损表面光滑,软骨陷窝明显,与周围正常软骨组织无明显界限;对照组为纤维组织修复。结论自体BMMSC诱导的软骨细胞可用于修复兔耳廓软骨缺损。     

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.     

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。     

同种异体软骨修复甲状软骨缺损的实验研究

OBJECTIVE: To study the effect of laryngeal cartilage defect repair with differently preserved allogeneic cartilage grafts. METHODS: 16 New Zealand white rabbits were used and divided into two groups. A 6 mm x 3 mm x 1 mm whole thickness cartilage defect was made in each side of the thyroid cartilage of each rabbit. In group one, the tissue-cultured cartilage grafts, preserved in RPMI-1640 medium for 30 days, were implanted in the left defects and the 4% formaldehyde preserved cartilage grafts for 30 days were implanted in the right defects. Fresh autogenous and allogeneic grafts were seprately transplanted into the right and left thyroid cartilage defects of the other group. Thyroid cartilages were taken out at 7, 30, 180 and 360 days after implantation. Samples were observed by macroscopy and prepared for hematoxylin and eosin (H&E) staining and immunohistochemical examination. RESULTS: No marked changes in form and volume were found in the fresh allografts and RPMI-1640 cultured cartilage grafts. The same as the autogenous cartilages, the defects of thyroid cartilage were successfully repaired by the fresh allografts and RPMI-1640 cultured cartilages. Whereas 4% formaldehyde preserved cartilage grafts were completely absorbed and replaced by cicatricial tissues in the thyroid cartilage defects. Histological observation showed that severe inflammatory cellular infiltration in the formaldehyde preserved cartilages at 7 and 30 days. The cartilage matrixes were resorpted and the chondrocytes showed degenerative change. Finally they were replaced by fibrous connective tissue at 360 days. In the RPMI-1640 cultured and fresh allogeneic cartilages only a little inflammatory cellular infiltration was observed and the cartilage matrixes were almost normal from 7 to 360 days. CONCLUSION: It is clinically feasible to use allogeneic grafts stored in RPMI-1640 medium or fresh allogeneic cartilage grafts for repairing laryngeal cartilage defects.     

汶川地震后部分地区耳鼻咽喉头面部伤情调查与分析

目的 调查分析强烈地震灾害造成的耳鼻咽喉头面部的伤情及救治.方法 对2008年5月12日四川汶川地震后第二军医大学医疗队所在安县、茂县、江油县及汶川县三江镇收治的、及巡诊的受伤人数进行调查,重点对伤员的耳鼻咽喉、颌面及头颈部的伤情、致伤原因、救治方法进行分析.结果 本组患者为地震3 d后存活人群中的耳鼻咽喉头面部伤227例,多是轻伤或伴有复合伤,其中185例头面部软组织伤,13例鼻骨或鼻窦骨折,18例鼻出血,7例颅底骨折,4例耳廓撕裂伤.根据救护所及医院的不同医疗条件进行诊断和治疗,对软组织损伤采用清创换药缝合,骨折尽可能复位.对伴有复合伤的危重伤员急救稳定后送至有条件的医院进一步治疗.救护所救治的46例中除3例复合伤或可疑视神经损伤的重伤员后送外,43例5～10 d痊愈.医院救治181例中除3例有颅底骨折的复合伤转院后送外,住院31例至5月26日痊愈出院26例,留院治疗的5例均为复合伤未愈者;147例未留院患者中除1例耳廓撕裂伤感染再次清创延期愈合外,146例软组织伤痊愈并拆,骨折患者在愈合中,未见并发症.结论 对地震后的耳鼻咽喉头面部伤应尽早清创缝合、骨折复位,注意合理用药,危重伤员及时后送,可以减少并发症的发生,降低死亡率.     

正常耳与聋耳电诱发听性脑干反应实验动物的研究

目的 通过耳蜗外电刺激探讨正常听力与致聋家猫耳蜗EABR检测效果的差异性.方法 选取听力正常家猫,行蜗外EABR检测,刺激电极置于圆窗龛6点和12点方向,分别记录EABR;联合应用卡那霉素和利尿酸钠制备重度耳聋实验动物模型,行蜗外圆窗龛6点方向的EABR检测,比较不同耳蜗外位点及致聋前后EABR的差异.结果 圆窗龛6点方向时EABR阈值为529+27μA,Ⅲ波及Ⅳ波潜伏期为2.16+0.12ms,2.91+0.14ms,12点方向时阈值为545+44μA,Ⅲ波及Ⅳ波潜伏期为2.18±0.13ms,2.93±0.10ms,两者无统计学差异(P＞0.05);致聋前EABR阈值为525+23μA,Ⅲ波及Ⅳ波潜伏期为2.16±0.11ms,2.89±0.14ms,致聋后EABR阈值为558+37μA,Ⅲ波及Ⅳ波潜伏期为2.17±0.09ms,2.91±0.12ms,致聋前后阈值及波潜伏期相比均无统计学差异.结论 刺激电极位点位于圆窗龛6点和12点方向时,EABR阈值及Ⅲ、Ⅳ波潜伏期无统计学差异,但6点方向时波形更容易引出;聋耳EABR波形形态与正常耳EABR形态相似,致聋前与致聋后EABR阈值、Ⅲ波及Ⅳ波潜伏期相比均无统计学差异.     

游离空肠重建气管对气道黏液清除功能影响的实验研究

目的　探讨游离空肠重建长段环形缺损气管后对气管的黏液 纤毛清除功能的影响 ,为该方法应用于临床提供依据。方法　通过显微外科游离空肠联合镍钛合金网重建 12条犬气管的动物实验模型 ,纤维支气管镜下于重建气道的下切缘用注射器滴入示踪剂 ,计算示踪剂从滴入到到达声门的时间 (mucociliarytransittime ,MTT)除该段长度 (mucociliarytransportlength ,MTL) ,为黏液清除率(mucociliarytransportrate,MTR) ,对游离空肠重建 6 5cm长段环形缺损气管后新气道的黏液 纤毛清除功能进行术前 ,术后 7d、1个月、3个月、6个月多时间点研究。结果　除了术前的MTR与术后 1个月的MTR相比差异有统计学意义外 (P <0 0 5 ) ,其余术前MTR与术后 7d、3个月、6个月及术后 7d与术后 1个月、3个月、6个月相比均无统计学意义 (P >0 0 5 )。结论　游离空肠重建气管中 ,新气道的黏液 纤毛清除功能随着呼吸道炎症的消退术后 3个月恢复正常水平     

人羊膜上皮细胞移植用于兔损伤声带组织修复的初步研究

目的 研究人羊膜上皮细胞(human amniotic epithelial cells,hAEC)移植入兔声带损伤组织内的生长分布特点,探索hAEC促进声带损伤后修复再生的潜能.方法 分离和培养hAEC,慢病毒增强型绿色荧光蛋白(1entivirus enhanced green fluorescent protein,Lenti-EGFP)基因转染作标记.建立深及声韧带的兔声带损伤模型,设hAEC移植组(13侧声带)、损伤对照组(13侧声带)及正常对照组(4侧声带).荧光显微镜下连续观察hAEC在声带内的存活、分布情况,应用HE染色和免疫组化染色分析胶原、纤维连接蛋白等主要细胞外基质在损伤后3个月时的含量及分布.结果 hAEC原代培养6 d后呈铺路石样生长,植入声带损伤组织后可在固有层内持续存活2个月,细胞呈纵向排列,有趋向性.声带损伤2个月时免疫荧光显示hAEC移植组兔肌细胞标志结蛋白荧光阳性,提示hAEC向肌细胞分化;同时Ⅲ型胶原荧光阳性,提示hAEC植入后具有分泌Ⅲ型胶原功能.光镜观察见hAEC移植后3个月兔胶原纤维密度和排序较损伤对照组改善,但未及正常;免疫组化染色示hAEC移植组纤维连接蛋白含量和分布介于损伤对照组和正常对照组之间.结论 hAEC可在异种动物声带损伤组织内持续存活、生长,并有向声带组织分化和分泌部分细胞外基质的潜能,可能促进声带损伤后的修复再生.

Abstract：

Objective To investigate the survival, growth and distribution of human amniotic epithelial cells (hAEC) after injected into injured rabbit vocal folds, in addition, to assess the ability of hAEC to affect the components of lamina propria extracellular matrix (ECM) and prevent vocal fold scarring.Methods hAEC were isolated from human amnion and marked by Lenti-EGFP. Fifteen New Zealand rabbits were used for this experiment. EGFP-hAEC was injected into the left injured vocal folds in thirteen rabbits, and the contralateral thirteen vocal folds experienced an injured procedure only (" injured untreated control"), and four vocal folds were left as untreated controls. The survival, distribution, differentiation potential and secretion function of hAEC were examined by immunofluorescence method. HE staining and immunohistochemical staining were performed for the evaluation of collagen and fibronectin respectively.Results hAEC showed a cobblestone-like growth. After implanted into the injured vocal folds, hAEC could survive in vocal fold lamina propria for 2 months. The immunofluorescence analysis showed the evidence of hAEC differentiation into muscle cells as well as secretion the ECM protein. Three months postoperatively, the density of collagen was higher in the injured untreated control folds than that in the injured vocal folds injected with hAEC and the untreated controls. Besides, the content of fibronectin in the injured untreated control group was significantly increased. Conclusions hAEC survived in the vocal folds lamina propria,and had the potentiality to differentiate into vocal folds tissue and secret some ECM components. The histological improvement caused by the injected cells demonstrate that hAEC had the ability to promote the repairment and regeneration of injured vocal folds.     

游离空肠段肠腺分泌规律及病理学变化

目的探讨游离空肠段肠腺分泌规律及其相应的病理学变化,为临床应用于气管重建提供依据。方法通过建立腹壁下去神经游离空肠的肠分泌动物实验模型,包括A组(无支架组)、B组(游离空肠联合镍钛合金内、外支架组)及C组(游离空肠联合镍钛合金外支架组),对比其术后肠分泌量的变化以及相应的病理学变化。结果①A、B、C三组术后早期肠分泌量增高,1月后肠分泌量显著减少并趋于稳定;B组和C组均比A组的分泌量大(P<0.05),1月后逐渐接近A组;B、C两组对比肠分泌量无显著差异(P>0.05);②移植空肠上皮腺体萎缩,肠黏膜上皮层变薄,微绒毛大部分脱落,部分吸收细胞空泡变性和坏死,杯状细胞减少;其中以B组改变最明显。结论去外源神经犬空肠段随着肠腺萎缩,肠腺分泌逐渐减少,术后60天左右趋于低水平;推断游离空肠重建气管是可行的。     

Biocompatibility of vitallium as ossicular reconstruction material in the middle ear: experimental animal study

Conclusion Although long-term data will be necessary for confirmation, the result of this preliminary study indicates that vitallium may be a good alternative material for ossicular replacement prostheses in the middle ear.

Objectives To investigate the biocompatibility of vitallium (Co–Cr–Mo) as ossicular reconstruction material in the rabbit middle ear, and to compare the results with those obtained with titanium, well known as a highly biocompatible material, and non-implanted control groups.

Material and methods Eighteen female New Zealand White rabbits were anesthetized. The tympanomeatal flap was elevated and 12 vitallium and 12 titanium implants were placed in the bulla away from the ossicles in 24 middle ears. Six rabbits were used as non-implanted controls. All animals were sacrificed under general anesthesia on the 180th day after implantation. The temporal bones were removed, fixed in 10% buffered paraformaldehyde and decalcified for a week in EDTA. Tissue samples were then prepared using an Autotechnicon and embedded in paraffin. Sections (30-μm thick) were cut with a microtome, stained with hematoxylin–eosin, von Gieson's stain and fibroblast growth factor (FGF) and examined under a light microscope. The numbers of lymphocytes, collagen fibers and FGF-positive cells were determined in all three groups.

Results There was no significant difference in the numbers of collagen fibers between the groups (p>0.05). No significant differences were found in the numbers of lymphocytes and FGF-positive cells between the titanium and vitallium groups (p>0.05). The differences in the numbers of lymphocytes and FGF-positive cells between the control and other groups were found to be significant (p<0.05).