组织工程化缓释骨形态发生蛋白2听骨赝复物听泡植入对豚鼠耳蜗功能的影响

目的 制备含缓释骨形态发生蛋白2(bone morphogenetic protein 2,BMP-2)的组织工程化听骨赝复物,植入豚鼠听泡内,观察其成骨诱导情况及对耳蜗功能的影响.方法 制备脱细胞骨微管,管腔内填塞含BMP-2胶原海绵,形成小柱状听骨赝复物.耳后人路植入豚鼠听泡,对侧耳植入不含BMP-2的赝复物作为对照.植入后不同时间点进行听性脑干反应(ABR)检测,观察听骨赝复物植入对豚鼠反应阈的影响.植入3个月后通过听泡组织切片及基底膜铺片,观察植入物成骨情况以及对听泡、耳蜗大体结构和毛细胞形态的影响.结果 术后动物健康状况良好.植入听骨赝复物即刻,ABR反应阈(声压级,x±s,下同)与术前[(15.5±2.8)dB]相比略有升高[(28.3±4.8)dB,P<0.05],3个月后已恢复正常[(16.1±4.0)dB,P>0.05].术后3个月,大体观察见植入的听骨赝复物被覆黏膜,耳蜗及听泡大体形态正常.组织学检查可见含缓释BMP-2的骨微管内壁有新骨形成,未见耳蜗及听泡骨质异常增生,镫骨底板与前庭窗周围骨质无融合.基底膜铺片硝酸银染色示毛细胞形态正常,未见缺失.结论 含缓释BMP-2的组织工程化听骨赝复物植入听泡后,可诱导新生骨形成,但对听泡及耳蜗形态结构无影响,不会引起周围骨质的异常增生,同时对ABR反应阈无影响.该方法有希望应用于听骨链缺损的修复重建.     

原位组织工程构建兔听小骨的研究

目的将猪小柱状脱细胞松质骨与骨形态发生蛋白2（bone morphogenetic proteins 2，BMP-2）复合制成听骨赝复物，植入兔听泡内，观察其原位成骨状况。方法制备猪小柱状脱细胞松质骨，与BMP-2复合作为听骨赝复物。选择5只新西兰大耳白兔，采用耳后进路，听泡后外侧骨壁钻孔，清除听泡内部分黏膜组织，一侧植入复合BMP-2听骨赝复物，对侧耳植入单纯脱细胞骨小柱作为对照。术后3个月，通过大体及病理切片观察听骨赝复物成骨情况。结果术后5只兔健康状况良好。术后3个月，植入的复合BMP-2脱细胞松质骨与听泡骨壁接触部位结合紧密，表面被再生黏膜覆盖，病理学检查发现有新骨形成，而对照耳仅有黏膜覆盖，无新骨形成。结论复合BMP-2的猪脱细胞松质骨可于兔听泡内形成新生骨组织，有可能用于听骨链缺损重建。     

耳蜗外毛细胞静纤毛束变异与听功能改变的关系

目的　探讨耳蜗毛细胞静纤毛束变异与听功能改变的关系。方法　测定 2 0只豚鼠听性脑干反应(ABR)阈和畸变产物耳声发射 (DPOAE)振幅 ,观察耳蜗毛细胞静纤毛变异形态 ,计数变异的毛细胞数目。结果　耳蜗毛细胞静纤毛束变异主要为第一排外毛细胞静纤毛转位 ,多呈 90° ,少数达 180°,两臂长短不一 ,“W”型夹角变大。在这些动物中 ,ABR反应阈变化不明显 ,DPOAE振幅显著增大。结论　豚鼠耳蜗外毛细胞静纤毛束变异具有普遍性 ,变异者伴有一定的听功能改变 ,DPOAE可作为判断变异的指标。     

模拟失重条件下飞船内噪声对豚鼠耳蜗形态与功能的影响

目的探讨模拟失重条件下,飞船内稳态噪声对豚鼠耳蜗形态与功能的影响。方法32只豚鼠随机分为单纯失重组16只、失重＋稳态噪声组16只。后肢悬吊法模拟失重,暴露于模拟飞船内在天飞行段的噪声环境,共5天。实验前、实验结束后即刻和实验结束后3天测试脑干诱发电位（ABR）阈值,取耳蜗标本行扫描电镜和透射电镜观察。结果实验组实验结束后即刻ABR阈值较实验前及实验结束后3天均增高（P〈0.01）;实验结束后3天ABR阈值较实验前高（P〈0.05）;实验结束后即刻及实验结束后3天失重＋稳态噪声组ABR阈值均较单纯失重组高（P〈0.01）。扫描电镜观察实验组实验结束后即刻耳蜗内、外毛细胞均受损。实验结束后3天,单纯失重组少数耳蜗各回内、外毛细胞的损伤程度比实验结束即刻加重;失重＋稳态噪声组耳蜗各回内毛细胞损伤较实验结束即刻重,外毛细胞损伤较实验结束即刻轻。实验组各时间段内毛细胞的损伤均重于外毛细胞,自第一回至第四回毛细胞损伤逐渐加重。透射电镜观察实验组耳蜗毛细胞及神经节细胞均可见空泡样改变,线粒体分布减少,细胞核固缩,可见细胞凋亡和细胞坏死两种细胞死亡现象。结论失重及失重＋稳态噪声均可造成豚鼠耳蜗形态和功能损伤,后者造成的损伤更重。失重对耳蜗毛细胞损伤以内毛细胞为重,损伤从底回至顶回逐渐加重。实验结束后3天较实验后即刻的听功能有所恢复但内毛细胞损伤加重。     

复方丹参抗耳蜗外毛细胞凋亡的实验观察

目的 探讨氨基甙类抗生素所致耳蜗外毛细胞凋亡与听力损害关系及复方丹参对其凋亡发生的影响。方法 对35只豚鼠行药物造模,测试ABR阈值,TUNEL技术观察耳蜗外毛细胞凋亡发生情况。结果 正常豚鼠耳蜗外毛细胞无凋亡发生,耳毒性药物应用1d,3d后耳蜗各转外毛细胞均可出现凋亡阳性染色,中药干预组动物耳蜗外毛细胞凋亡数目相对较少,此外,豚鼠ABR阈值皆无明显升高。结论 耳毒性药物可诱发耳蜗外毛细胞发生凋亡;复方丹参能够对抗凋亡发生;凋亡发生早期对豚鼠听力阈值无明显影响。     

脱细胞骨微管用于构建组织工程化听骨赝复物的可行性

目的观察脱细胞骨微管用于制备组织工程化听骨的可能性。方法制备脱细胞骨微管,观察其组织相容性及生物力学变化,并作为含复合骨形态发生蛋白2(bone morphogenetic protein,BMP-2)胶原海绵载体,植入动物中耳空腔,进行异位诱导成骨观察。结果脱细胞骨微管具有良好的生物相容性,其生物力学性能无显著变化。负载含BMP-2胶原海绵后,可以在中耳腔内诱导形成新生骨组织。结论脱细胞骨微管负载含BMP-2胶原海绵可用于构建组织工程化听骨赝复物。     

盐酸椒苯酮胺对豚鼠耳蜗缺血再灌注损伤的听力保护作用北大核心CSCD

目的探讨盐酸椒苯酮胺(piperphentonamine hydrochloride,PPTA)对豚鼠耳蜗缺血再灌注损伤后听功能保护作用及其对耳蜗组织形态的影响。方法将32只成年豚鼠随机分为4组,每组8只,第1组为正常组(不做任何处理),第2组为空白对照组(手术切开颈前正中皮肤,分离出双侧椎动脉、双侧颈总动脉,但不做其它处理),第3组为缺血再灌注对照组(阻断双侧椎动脉及右侧颈总动脉建立豚鼠耳蜗缺血再灌注模型,再灌注同时股静脉给予与PPTA同等剂量生理盐水),第4组为缺血再灌注实验组(手术造模成功后,再灌注同时静脉给予PPTA 10mg/kg)。测量实验前后各组动物的听性脑干反应(ABR)波Ⅲ反应阈。动物造模成功后24小时迅速断头取听泡,每组取4只扫描电镜下观察耳蜗结构、另外4只用透射电镜观察耳蜗结构。结果实验前4组动物ABR波Ⅲ反应阈差异无统计学意义(P>0.05),缺血再灌注24h后第3组ABR波Ⅲ反应阈显著高于第1组和第2组(P<0.05),第4组ABR波Ⅲ反应阈比第3组明显降低(P<0.05);扫描及透射电镜下可见第3组耳蜗外毛细胞纤毛倒伏、脱落,内毛细胞纤毛散乱,血管纹内皮细胞核固缩、边集,神经元可见脱髓鞘改变,而第4组的耳蜗组织损伤明显减轻。结论 PPTA可以防止耳蜗缺血再灌注损伤后毛细胞损伤缺失,对豚鼠耳蜗缺血再灌注听力损伤有保护作用。     

碱性成纤维细胞生长因子脑脊液途径用药对听觉系统保护作用的实验研究

目的探讨碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）脑脊液途径用药对硫酸丁胺卡那霉素诱导的豚鼠听觉损害的防治作用,为临床有效防治感音神经性聋提供理论依据。方法应用硫酸丁胺卡那霉素肌肉注射诱导豚鼠听觉损害,将bFGF采用脑脊液途径注入豚鼠体内。用药结束后测试听性脑干反应（auditory brainstem response,ABR）阈值,行耳蜗灌注琥珀酸脱氢酶（succinate dehydrogenase,SDH）组化染色,耳蜗铺片,观察染色强度及内、外毛细胞损伤程度,取脑干和双侧耳蜗行甲苯胺蓝染色,耳蜗螺旋神经节细胞计数,并测量相应的积分光密度（integral optical density,IOD）。结果用药后各组动物ABR阈值测试有显著性差异;耳蜗SDH染色和铺片内、外毛细胞损伤程度不同;耳蜗切片螺旋神经节细胞计数及IOD测定、耳蜗核切片IOD测试各组间比较有显著性差异。结论bFGF脑脊液途径用药对硫酸丁胺卡那霉素诱导的豚鼠听觉系统损害有防治作用。     

耳蜗外毛细胞静纤毛束变异的判定标准与抵抗耳中毒的能力

目的建立耳蜗外毛细胞(OHC)静纤毛束变异的判定标准,观察毛细胞静纤毛束变异对庆大霉素耳中毒的抵抗能力.方法对豚鼠测定听性脑干反应(ABR)阈值和畸变产物耳声发射(DPOAE)振幅后,选取静纤毛束正常和变异豚鼠各5只,连续肌注庆大霉素(grntamicin,GM)12 d后,测定ABR阈值,扫描电镜观察耳蜗外毛细胞静纤毛束变异情况,耳蜗铺片计数毛细胞的损失数.结果耳蜗底回第一排外毛细胞静纤毛束转位超过45°、"W"变形数超过10%,作为豚鼠耳蜗外毛细胞静纤毛束变异的判定标准.5只静纤毛变异豚鼠GM注射12 d后平均ABR阈值51.0±8.76 dB SPL,外毛细胞损失约16%～39.96%,柯替器受损程度较轻;而外毛细胞(OHC)正常豚鼠GM注射12 d后ABR阈值上升到58.4～100 dB SPL,OHC基本消失,柯替器毛细胞破坏较为严重.结论耳蜗外毛细胞静纤毛束变异豚鼠较OHC正常者对GM中毒具有较大的耐受能力.     

肾阴虚对豚鼠听力的影响及补肾中药的拮抗作用

目的：探讨肾阴虚对豚鼠听力的影响及补肾中药对这一影响的拮抗作用,为进一步研究肾与耳的关系提供新的实验资料,方法,利用糖皮质激素模拟豚鼠肾阴虚模型,应用其廓反射（PR）及听性脑干反应（ABR）测试动物的听力变化,采用改良耳蜗铺片及硝酸银染色法在光镜下进行耳蜗毛细胞计数,结果：肾阴虚豚鼠表现为高频听力下降,尤以6kHz,为明显,ABRI,Ⅱ波潜伏期延长,而Ⅰ-Ⅱ波间期无变化,耳蜗第1－3回外毛细胞轻度坏死,尤以底回较明显,滋补肾有的中药对这一影响具有拮抗作用,而温补肾阳的中药作用不明显。结论：肾阴虚对豚鼠听力的影响主要是以6kHz为中心的高频听力下降,其原因可能与肾阴虚引起耳蜗底回外毛细胞部分坏死有关,应用补肾法治疗耳聋必须辨证使用滋补肾阴与温补肾阳两类中药。     

A study on reconstruction of ossicular chain by an in situ bone tissue engineering technique

Conclusion. The acellular cancellous bone with recombinant bone morphogenetic proteins 2 (rhBMP-2) could induce osteogenesis in the acoustic vesicle; this material might be used to reconstruct defects of the ossicular chain. Objective. In recent years, the in situ tissue engineering technique has improved rapidly, especially in bone regeneration. The aim of this study was to make an ossicular prothesis using columnar acellular cancellous bone combined with rhBMP-2, implant this prosthesis into the acoustic vesicle of rabbits, and then observe the osteogenesis in situ. Materials and methods. We prepared the acellular cancellous bone combined with rhBMP-2 as an ossicular prothesis, while the same material without rhBMP-2 was used in the control group. From the retroauricular approach, we made a hole in the posterolateral bone wall of the acoustic vesicle, and the prepared materials were implanted. After 3 months, we observed the osteogenesis of the prothesis by macroscopic anatomic and histologic methods. Results. The rabbits recovered soon after surgery. The implanted acellular cancellous bones were connected tightly with the bone of the acoustic vesicle wall. The surfaces of all materials were covered with mucosa, and osteogenesis was observed in the material with rhBMP-2.     

A morphometric study of the effects of pressure on bone resorption in the middle ear of rats

Bone destruction is one of the clinical features of chronic otitis media with cholesteatoma. Bone resorption may be due to the epithelial debris accumulated in the cholesteatoma epidermal sac that acts as foreign body material inducing a destructive granulation tissue and creates pressure on the bony middle ear. This compressive force then induces bone resorption. The present study was designed to replicate certain conditions similar to those in cholesteatoma leading to bone resorption. Four types of materials were implanted into the middle ear cavity of rats: (1) laminaria, an expandable seaweed material, (2) preswollen laminaria, (3) keratin powder suspension, and (4) surgical grade silicone, which when bent exerts pressure on the bulla wall. The placement of laminaria segments in the middle ear cavity of rats was followed by swelling of the implanted materials within 7 days. The bulla bone response was by neo-osteogenesis as well as active bone resorption. The new bone was observed on the external and/or internal surface of the tympanic bone. The cochlear bone also showed extensive bone resorption in the animals. Osteogenesis was rarely observed on the capsule of the cochlea. We also observed no bone resorption at sites without presence of inflammatory connective tissue between laminaria and bone. The typical multinucleated osteoclasts were often seen at the resorption area but the majority of bone resorption sites were characterized by the presence of mononuclear cells and other inflammatory cells. Preswollen laminaria, keratin powder, and silicone strips induced minimal bone resorption. No resorption was observed in the bony cochleas of these experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the osteogenic activity of bone morphogenetic protein (BMP) mutants]

BACKGROUND: Modification of the heparin binding site by alteration of the amino acid sequence of bone morphogenetic protein-2 (BMP-2) results in a change in the local retention time. The purpose of this study was to compare the osteogenic activity of T3 and T4, two mutants with increased binding capacity to heparin, and B2GDF-5 a mutant resulting from the fusion of the n-terminal amino acid sequence of BMP-2 and the c-terminal sequence of GDF-5 with wild-type BMP-2 in vivo. MATERIAL AND METHODS: The proteins were coupled to an equine-derived collagen carrier and implanted in standardized critical size calvarial defects in adult rats. After 28 days, bone formation was evaluated radiographically and the new bone was characterized histologically. RESULTS: Proteins T3 and T4 showed a higher osteogenic activity than BMP-2. Less new bone formation was observed with GDF-5 and B2GDF-5 than with-type BMP-2. No difference in bone formation was observed between GDF-5 and B2GDF-5. CONCLUSION: Increased heparin binding capacity enhances osteogenic activity of BMP-2 in vivo. This might be due to a longer retention period in the tissue and thus better bioavailability. Replacement of the N-terminal amino acid sequence of GDF-5 by the corresponding sequence of BMP-2 did not result in an increased osteogenic activity as heparin binding capacity is not the main reason for the bioavailability of GDF-5.     

Mastoid obliteration by BMP-2/collagen composites: an experimental study using tissue engineering

PURPOSE: Several materials have been used in the application of mastoid cavity obliteration during surgery for cholesteatoma; however, nothing has won universal acceptance. Through the advancement of tissue engineering, bone morphogenetic protein-2 (BMP-2)/collagen composites have been elucidated as inducers of heterogenic bone formation. This study was performed to investigate whether these composites are potentially obliteration materials for use in the mastoid cavity by using an animal experimental study. MATERIALS AND METHODS: The composites were implanted in the rat mastoid to investigate whether new bone would be tissue engineered in the mastoid and, if so, whether the newly formed bone was stable. The composites were examined histologically over a 24-week period. RESULTS: The composites implanted in the rat mastoid were able to tissue engineer new bone, and the newly formed bone was stable as assessed histologically, with almost normal bone structure, that was not resorbed during the 24-week period. Adverse immunological reactions were not found during our observation. CONCLUSIONS: Bone that was tissue engineered by the BMP-2/collagen composites was stable as assessed by histological examination and persisted in the rat mastoid. The present study shows that the composites have the potential to become real materials for use in mastoid obliteration.     

Osteoclast stimulation by positive middle-ear air pressure

The mechanism of pathologic bone resorption associated with cholesteatoma is controversial. It is now evident that transmitted pressure is a significant factor in the activation of osteoclasts. Previous studies on the effect of pressure have been confounded by the possible foreign body effect of materials placed into the middle ear to induce the pressure. Positive air pressures were maintained in the middle ear of Mongolian gerbils via implanted Silastic catheters. Histologic evaluation of the ventral bulla wall was performed after pressure had been applied for two weeks. A significant increase in the number density, area density, and size of osteoclasts present in the pressurized bullae was noted at pressures of 4, 6, 10, and 20 mm Hg, but not at 2 mm Hg. We conclude that minute increases in pressure, in the absence of foreign objects, stimulate osteoclastic bone resorption in the middle ear of the gerbil.     

The effect of chronic middle ear inflammation on the pneumatization of the tympanic bulla in pigs

The effect of chronic middle ear inflammation on the pneumatization of the tympanic bulla was investigated in piglets. The pig tympanic bulla has an air cell system which is divided by trabeculae and closely resembles the human mastoid air cell system. The tympanic bulla and its air cell system in normal ears were well developed because of the bone formation and the bone resorption inside the cortex, whereas the tympanic bulla affected by chronic otitis media in the early stages of life exhibited retardation of pneumatization arising from the disturbed bone resorption by inflammatory stimulus. It was concluded that affliction with chronic middle ear inflammation in the early stages of life causes inhibition of pneumatization by hindering the development of the air cell system.     

Experimental pilot study on surface activation of implants with liposomal vectors]

BACKGROUND: Surface coating with mitogenic or morphogenic proteins can improve the healing of bone adjacent to implants and increase the bone-implant interface. Clinical surveys have shown liposome-mediated gene transfer to be a promising and safe new therapeutic method. The aim of our study was to evaluate an experimental model of new approaches for topical treatment of the implant surface and of periimplant defects by using DNA liposomes encoding for BMP-2 (bone morphogenetic protein). MATERIAL AND METHODS: A total of 27 implants (3.5 x 14 mm) were placed in critically sized defects of the frontal skull bone of adult pigs (n=3). The bottom of the implant was placed in the base of the defect which guaranteed primary stability, whereas the superior part of the implant (10 mm) represented an implant in a defect area. Liposomes containing DNA encoding for BMP-2 and GFP (green fluorescence protein) were used. In a first trial GFP-DNA liposomes on a collagen matrix were directly applied to the periimplant defect. In a second stage, the surface of the implants was encoded with BMP-2 DNA liposomes. Subsequently, these implants were inserted in the manner described. The resulting bone samples were prepared for immunohistochemical staining. Staining for GFP was performed in the area of the defect and for BMP-2 on the bone-implant interface. RESULTS: Immunohistochemical staining on day 3 postoperatively revealed an increased GFP expression in the periimplant defect. Therefore, the effectiveness of the liposomal vector was verified for the chosen animal model. On the surface of the implants encoded with BMP-2 DNA liposomes an increased BMP-2 expression was found. Thus, the liposomal vector system was validated also for BMP-2 DNA transfer in the chosen animal model. Further, the established system allows a sustainable and delayed release of BMP-2 in the area of the bone-implant interface. CONCLUSIONS: As a result of the study we were able to collect data concerning the influence of implant surface conditioning on the bone-implant interface and on therapeutically relevant options for the treatment of periimplant defects. These approaches are currently being evaluated in a long-term study.