液氮冷冻致兔股骨头坏死病理学特征研究

目的 探讨液氮冷冻法制备兔股骨头坏死动物模型的病理学特征.方法 采用成年新西兰大白兔,无菌条件下手术显露股骨头;采用棉签蘸取液氮,对股骨头软骨面负重区冷冻、复温以制备股骨头坏死模型.于制模后第3、7天及第2、4、6、8周随机选择模型兔,手术取下股骨头,进行股骨头纵切面肉眼观察,常规石蜡包埋、切片,经HE染色后光镜观察.结果 股骨头纵切面4周前各观察时间点,股骨头局灶性坏死区域渐增.制模术后第3天兔股骨头组织切片镜下可见软骨细胞及骨细胞坏死;术后第7天见坏死区空骨陷窝较多;术后第2周见关节软骨缺损,软骨下区域小血管充血,骨小梁开始断裂、排列紊乱;术后第4周见关节软骨剥脱,髓腔内脂肪细胞坏死,并现增生纤维组织;术后第6周见股骨头塌陷,并出现纤维化骨的匍伏附着;术后第8周见新骨沉积性生长,骺板细胞挤压、变形.结论 本研究建立的液氮冷冻致兔股骨头坏死模型具有典型的病理演变特征,贴近已知人类股骨头坏死病理演变规律,是干细胞移植等治疗研究的理想模型.     

高强度聚焦超声制备兔股骨头坏死模型的可行性研究

目的:探讨高强度聚焦超声新方法制备兔股骨头坏死模型的可行性。方法:采用JC200型聚焦超声治疗系统,以80 w/cm~2强度超声照射家兔双侧股骨头,分别于照射后1、7、14、21 d取材,结合股骨头大体形态和组织石蜡切片H-E染色观察股骨头坏死情况。结果:股骨头外观7 d时可见界限明显的白色坏死区,表面光泽度稍下降;14 d时坏死区表面软骨变黄、皱缩;21 d时囊化、易剥脱。镜下见1 d时坏死区表面软骨可见少量软骨细胞核溶解,髓质可见小静脉扩张充血、血窦充血;7 d时坏死区表面软骨轻微挤压皱缩,软骨下区见骨小粱断裂和空骨陷窝,髓质造血组织明显减少,骨小梁表面成骨细胞消失,坏死骨小梁附近增生纤维组织,出现脂肪小囊,骺板稍变形;14 d时兔股骨头表面软骨变薄,偶可见破损,坏死软骨细胞增多,表面破损程度、骺板变形程度较7 d时加重,修复区可见增生肉芽组织和新生血管,有纤维组织附着于坏死骨小梁;21 d时,股骨头表面软骨破损严重,骺板进一步扭曲变形,坏死区骨小梁破碎、消失,修复区可见纤维组织包绕坏死骨小梁和新生骨小梁。结论:高强度聚焦超声制备的兔股骨头坏死模型效果稳定,具有清晰的病理演变特征,可作为一种制备股骨头坏死模型的新方法。     

液氮冷冻法制作兔股骨头坏死模型:可行,理想与可信

背景:目前缺乏一种理想股骨头坏死动物模型,从而制约了对其进一步的研究。目的:验证以液氮冷冻法制作兔股骨头坏死修复模型的可行行,为后续研究奠定基础。方法:取成年新西兰大白兔20只,双侧进行实验,在不切断股骨头圆韧带、髋关节不脱位的基础上,以液氮棉签连续快速冷冻股骨头25次,约10s/次。分别于术后第3,7天,2,4,6,8周拍双侧髋关节正位片X射线片、MRI,并进行股骨头标本的大体解剖学观察和组织学观察。结果与结论:在造模3d时的股骨头组织切片上,冷冻区和周围部的软骨细胞、骨细胞和骨髓组织呈现坏死。2周,可见坏死的骨小梁周围开始出现修复表现。4周,部分出现剥脱,6周,软骨剥脱更加明显,8周,呈骨性关节炎改变,关节软骨剥脱严重,有2例股骨头正常外形改变。证实非髋关节脱位情况下液氮冷冻股骨头,可以成功建立的兔股骨头坏死模型,简便易行成功率高,可作为其后续研究的理想模型。     

重组合异种骨对兔股骨头缺血坏死模型实验修复过程的影响

目的 本文旨在探讨含骨形态发生蛋白(BMP)的重组合异种骨是否对液氮冷冻兔股骨头缺血坏死模型有促进修复作用。方法 本实验采用新西兰大白兔24只,雌雄各半,体重2kg。用液氮冷冻法造成左侧股骨头坏死,并且在同侧股骨颈后方头颈交界处钻直径2mm孔至软骨下,随机分成实验组和对照组各12只,实验组术中植入重组合异种骨(RBX)30mg/只(含BMP1.5mg),对照组术中植入同体积的牛松质骨,于术后2、4、8周分别处死实验组和对照组各4只动物,观察有关指标。结果(1)X线检查结合计算机相对骨密度分析:2周时两组股骨头骨密度均未见增高,4、8周时均增高,但手术侧实验组股骨头相对骨密度显著高于对照组(P<0.05)。(2)单光子骨密度(BMD)测定:4周以后手术侧实验组股骨头骨密度显著高于对照组(P<0.05)。(3)组织学观察:4周时实验组手术侧股骨头内纤维组织增生,有大量的软骨形成和成骨细胞增生明显,新骨形成多于对照组,8周时实验组手术侧股骨头内坏死骨组织修复大部分已完成,表面软骨再生。而对照组坏死骨修复仍在大部分区域内进行,植骨仍有部分未被吸收。结论 重组合异种骨对液氮冷冻兔股骨头坏死实验修复过程有明显的促进作用。     

三足负重犬股骨头坏死模型的生物力学相关性研究

目的研究股骨头坏死病程各阶段的生物力学改变,探讨其生物力学发生作用的机制。方法取杂种犬24只,固定一侧前肢建立三足负重犬模型。随机取三足负重犬一侧后肢为实验侧,在股骨头内注射无水酒精,致股骨头坏死;对侧为对照侧,在股骨头内注入等量生理盐水。造模后1、3、6及12周处死动物,每组6只,分别对两组股骨头行大体观察以及放射学、组织学、生物力学检测。结果术后3周股骨头坏死侧点压硬度及中部松质骨弹性模量相对对照侧分别下降29%和32.9%,此时仅MRI可见股骨头出现低密度坏死区,组织学主要表现为骨坏死。术后6、12周股骨头坏死侧点压硬度相对对照侧分别下降了45.5%和48.7%,中部松质骨弹性模量相对对照侧分别下降了34.1%和32.4%。6周时坏死侧X线可见股骨头内密度不均,组织学表现为坏死和修复反应并存,坏死区向软骨下骨区进展。12周时坏死侧X线可见股骨头负重区下出现局部骨密度减低区,组织学出现关节软骨面塌陷和关节间隙狭窄。结论生物力学是股骨头病程进展的重要影响因素;病程进展中,特别是修复期力学性能的显著下降可能是后期塌陷的最直接原因。治疗股骨头坏死不仅应促进骨修复,更应提供病变区一个有利、稳定的生物力学环境。     

普伐他汀干预激素性股骨头坏死兔模型的组织学变化:大体和光镜下比较和印证

背景:研究表明普伐他汀可通过上调内源性骨形成蛋白2、核心结合因子α1和血管内皮生长因子等基因的表达从而产生促进激素性股骨头坏死兔模型坏死股骨头修复的作用。目的:验证性观察普伐他汀干预激素性股骨头坏死兔模型大体和光镜下的组织学改变,并进行相互比较和印证。方法:将80只新西兰白兔按随机数字表法分为对照组18只,实验组62只。向实验组耳缘静脉注射大肠杆菌内毒素(10μg/kg)2次,注射间隔24h。并于第2次注射大肠杆菌内毒素后,臀肌注射甲强龙(20mg/kg)3次,注射间隔24h,制备兔激素性股骨头缺血坏死模型。造模第5周,将造模成功的39只新西兰白兔以随机数字表法分配其中的36只至模型组和他汀组,每组18只。他汀组以普伐他汀(1.2mg/kg)灌胃,1次/d;模型组和对照组以等体积的蒸馏水灌胃。造模后8,12,16周,取股骨头,分别进行大体和光镜观察。结果与结论:大体观察及光镜观察显示模型组出现明显的骨坏死灶,骨髓腔内可见大量脂肪细胞增生;与模型组比较,他汀组骨细胞受损程度较轻,骨小梁密度较高,空骨陷窝比率较少,骨坏死面积和髓腔内脂肪细胞数量明显减少,骨坏死修复迹象明显。说明普伐他汀可有效促进早期激素性股骨头坏死兔模型坏死股骨头的修复。     

富血小板血浆凝胶联合髓芯减压修复兔股骨头无菌性坏死

背景：近年来的研究表明,富血小板血浆有较强的成骨能力且在骨科及口腔科领域已得到广泛应用,而其在股骨头坏死方面的实验未见报道。 目的：观察富血小板血浆凝胶联合髓芯减压修复股骨头无菌性坏死模型兔的效果。 方法：取新西兰大白兔25只,在不造成髋关节脱位的基础上,用液氮冷冻兔双侧股骨头建立无菌性坏死模型,造模后2周,随机分组：模型组9只不做任何治疗,髓芯减压组8只行髓芯减压治疗,联合组8只采用富血小板血浆凝胶联合髓芯减压治疗,造模后第8周采集股骨头标本组织切片行MRI检查及苏木精-伊红染色。 结果与结论：①MRI检查：模型组表现为股骨头脂肪高信号中出现不同形态的低信号区,呈环形、带状和灶状;髓芯减压组减压缺损区无明显成骨改变,缺损腔无明显缩小,以长T1、T2信号为主;联合组缺损腔消失,被短T1、T2的大量新生骨所充填。②苏木精-伊红染色：模型组关节软骨缺损与修复同时存在,呈现骨性关节炎样改变;髓芯减压组骨小梁空骨陷窝,骨髓内造血细胞和脂肪细胞坏死;联合组骨小梁空骨陷窝,骨髓散在灶状坏死。表明富血小板血浆凝胶联合髓芯减压治疗股骨头坏死模型兔的效果优于单纯髓芯减压治疗。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

VEGF在高压氧治疗激素性股骨头坏死中的意义

目的:观察血管内皮生长因子(VEGF)在激素性股骨头坏死及高压氧治疗过程中的变化,探讨激素性股骨头坏死的发病机理。方法:健康日本成年大耳白兔60只,随机分为模型组(n=42)及对照组(n=18)。模型组每周两次肌肉注射醋酸泼尼松龙10mg/kg,对照组每周两次肌肉注射生理盐水2ml。共6周。随后将模型组随机分为高压氧治疗组(n=16)及常压常氧组(n=16),前者行高压氧治疗,后者呼吸常压新鲜空气,共持续6周。检测实验第2周、4周、6周、8周、10周、12周各组兔骨组织VEGF表达、组织病理学及影像学变化。结果:造模第6周时股骨头较多骨细胞坏死溶解成碎片,股骨头软骨下区骨小梁表面的成骨细胞和血管仅出现少量VEGF阳性表达;高压氧治疗第6周,实验兔骨小梁周围出现较稀疏的骨母细胞,电镜发现新生的骨细胞和胶原纤维,VEGF阳性细胞明显增多,软骨下阳性血管也明显增多,阳性部位主要位于血管内膜,同时在染色阴性区域出现点状新生毛细血管。结论:高压氧通过促进VEGF等细胞因子的分泌,加速股骨头的再血管化和再骨化进程,促进骨修复。     

医用TH胶栓塞建立月骨缺血性坏死兔模型

背景：目前缺乏与人类月骨缺血性坏死病理过程类似的Kienböck病动物模型。目的：采用医用TH胶栓塞法建立一种新型月骨缺血坏死兔模型,并探讨造模方法的合理性。方法：选择健康成年新西兰兔30只,雌雄不限。采用自身对照方法随机分为实验侧和对照侧,实验侧采用月骨中心钻孔,注射医用TH胶0.2 mL,反复注药3次;对照侧月骨中心钻孔注射同等量的生理盐水。于实验4,8,12周行X射线、大体观察、Micro-CT 骨计量学及组织病理学检测。结果与结论：大体标本、X射线及组织学检测结果可见实验侧造模后8周月骨出现缺血坏死表现,12周月骨缺血坏死更为典型。Micro-CT 骨小梁微观参数中,实验侧骨小梁密度参数骨体积分数及骨小梁数量均明显低于对照侧(P < 0.05);骨小梁空间参数明显增高,其中实验侧骨小梁离度及结构模型指数明显高于对照侧。提示注射医用TH胶栓塞后8周实验侧出现月骨缺血坏死表现,注药后12周可建立月骨缺血坏死兔模型,可以实现在周围组织无明显损伤情况下出现月骨内血运受损、骨小梁骨折、空骨陷窝等与人类月骨坏死类似的改变。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

BMP-2转染BMSCs复合镁合金棒修复兔股骨头坏死的实验研究

目的　探讨BMP-2转染BMSCs复合镁合金棒修复兔股骨头坏死的的效果。 方法　取一只兔分离、培养兔BMSCs,采用电转法将成功转染pcDNA3.1-BMP-2的质粒导入BMSCs。取40只兔子用液氮冷冻法制造股骨头坏死模型,然后随机分为4组,每组10只,分别为镁棒/BMSCs组,镁棒组,BMSCs组,空白组。镁棒组和镁棒/BMSCs组是指利用生物钻将生物镁合金棒置入坏死股骨头,同时镁棒/BMSCs组和BMSCs组导入转染pcDNA3.1-BMP-2质粒的BMSCs。术后6周和12周分别取股骨头标本观察修复效果。 结果　术后第1、2、4、8、12周分别检验各组兔血液、尿液中镁离子浓度。第6、12周X线、HE染色显示,镁棒/BMSCs组股骨头恢复最好,第12周镁合金棒基本完全吸收;镁棒组和BMSCs组股骨头坏死进展较慢,部分组股骨头坏死有好转;空白组股骨头坏死加重。 结论　植入镁合金棒联合BMP-2基因复合BMSCs有延缓和修复股骨头缺血性坏死的作用。     

IGF-1在股骨头再造关节软骨化生中的作用

目的观察分析大转子骨瓣表面骨膜及腱膜等纤维结缔组织向关节软骨转化的规律及胰岛素样生长因子-1(IGF-1)在软骨化生过程中的作用。方法制备液氮冷冻双侧股骨头缺血性坏死(Osteonecrosis of femoral head,ONFH)的动物模型。左侧股骨头造模后即缝合关节囊,右侧股骨头根据分组不同采用不同的处理方式:A组(骨瓣治疗组):带血管蒂大转子骨瓣进行股骨头再造;B组(骨瓣加Ad-IGF-1基因治疗治疗组):带血管蒂大转子骨瓣股骨头再造,关节内注入表达IGF-1的腺病毒载体(Ad-IGF-1);两组动物分别于3,6,12,18,24周每批4只处死,对骨瓣进行大体观察,组织病理学观察,免疫组化检测。结果所有动物左侧冷冻区组织坏死,纤维状物覆盖,碎片样组织修复。组织病理切片及免疫组化证实A组右侧骨瓣区自6周出现透明软骨细胞,B组右侧骨瓣区自3周出现透明软骨细胞,B组较A组修复效果好。结果经统计学处理有统计学意义。结论大转子表面的骨膜及腱膜能够向关节软骨化生,IGF-1对大转子表面的骨膜及腱膜向关节软骨化生有促进作用,为ONFH的外科治疗及生长因子的应用提供基础。     

骨内注射无水乙醇建立兔股骨头坏死模型

背景：目前还难以建立合适的早期股骨头坏死的动物模型。 目的：建立一个简单、标准和可靠的股骨头坏死动物模型用于实验研究。 方法：X射线透视下将新西兰兔右侧股骨头中心钻孔并注入无水乙醇,左侧不做处理做对照。经过2,4和6周麻醉下处死动物获取股骨头。 结果与结论：大体及X射线观察显示,造模侧股骨头第2周关节软骨颜色变暗、骨质密度不均匀;第6周,关节面有轻微凹陷,骨质低密度影较前进一步增大。MRI显示,造模侧股骨头第2周,T1加权股骨头负重区显示线样或不规则低信号,T2加权呈高信号;第6周,股骨头变性,软骨下骨折,关节面塌陷,新月体形成。对照侧第2,4,6周股骨头结果均常。组织病理学观察显示,造模侧股骨头第2周后,骨细胞核固缩、变性坏死。结果证实,2周后兔股骨头均发生了部分坏死,股骨头坏死的完整的外观形态,大体循环和关节软骨与人类早期股骨头坏死是相似的,提示实验建立了良好的股骨头坏死动物模型。     

构建双足类大型鸟类鸸鹋股骨头坏死模型

BACKGROUND: There are many studies on the establishment of animal models of femoral head necrosis, but it is difficult to simulate an ideal animal model of femoral head necrosis, especially collapse models. OBJECTIVE: To establish femoral head necrosis model of double-foot large bird emu so as to simulate femoral head necrosis in human.   METHODS: A total of 30 adult emus were frozen in liquid nitrogen and received radiofrequency heating alternately to simulate femoral head necrosis models. After 3 cycles of freezing, local frozen in liquid nitrogen and method of radiofrequency heating was alternated for closed loop liquid nitrogen freezing injury and radiofrequency heating injury. At 6, 12 and 16 weeks after model establishment, gross observation, X-ray, histology examination and MRI were conducted. Effects of model induction of avascular necrosis of the femoral head were evaluated.  RESULTS AND CONCLUSION: After liquid nitrogen cold-hot alternating method, emu at 12-18 weeks gradually suffered from femoral head necrosis signs and imaging findings. X-ray films, histological examination and MRI examination showed the avascular necrosis of femoral head was consistent with the pathological process of human femoral head necrosis, and the model had good repeatability. These results indicate that the use of liquid nitrogen cold-hot alternating method has successfully established the model of femoral head necrosis, with a good collapse rate, and can be used for mechanism research and treatment evaluation of osteonecrosis.      

骨髓间充质干细胞移植联合氯化锂治疗兔股骨头缺血坏死疗效

BACKGROUND: Lithium chloride can promote the proliferation and osteogenic capacity of bone marrow mesenchymal stem cells in the necrotic region after avascular necrosis of the femoral head, which has become an issue of concern. OBJECTIVE: To compare the advantages and disadvantages of bone marrow stem cell transplantation combined with lithium chloride in the treatment of rabbit femoral head necrosis. METHODS: Passage 2 bone marrow mesenchymal stem cells from 1-week-old New Zealand rabbits were cultured in 0, 5, 10, 20, 40 mmol/L lithium chloride. Forty-eight healthy adult New Zealand rabbits were selected to make femoral head necrosis models in the right femoral head using liquid nitrogen freezing method and then randomized into four groups: model group with no implantation; lithium chloride group given lithium chloride treatment at 3 days after modeling; cell transplantation group given gelatin sponge implantation and bone marrow mesenchymal stem cell suspension injection into the femoral head after modeling; combined group given bone marrow mesenchymal stem cell suspension injection and lithium chloride treatment. Intraperitoneal injection of lithium chloride (45.2 mg/kg) was given daily beginning at the postoperative 3rd day, and the treatment duration was 4 weeks. RESULTS AND CONCLUSION: Lithium chloride at 10 mmol/L had the maximum effect on the proliferation of rabbit bone marrow mesenchymal stem cells, and if the concentration of lithium chloride was > 10 mmol/L, the promotion role of lithium chloride began to decline. After combined treatment, the morphology of the femoral head was restored a little, with increased bone density and thickened trabecular bone; the level of β-catenin in the femoral head was significantly increased in the combined group compared with the cell transplantation group or the lithium chloride group. These findings show that bone marrow stem cell transplantation combined with lithium chloride treatment can promote the recovery from femoral head necrosis by increasing bone mass of the trabecular bone and bone density of the femoral head in the necrotic region.      

髓芯减压和骨髓基质细胞移植对家兔股骨头缺血性坏死的治疗作用

目的:探讨髓芯减压和体外培养的同种异体骨髓基质细胞移植,对兔股骨头缺血性坏死的治疗作用和修复机制。方法:采用新西兰大白兔液氮冷冻法造模,随机分为髓芯减压(A)组和同种异体细胞移植(B)组。术后标本做X-线和组织学观察。结果:(1)X-线观察2周时A、B组股骨头钻孔区边缘密度增加;8周时A组钻孔区呈低密度,边缘形成硬化线,B组钻孔区出现骨小梁结构。(2)组织学观察2周时A组钻孔区边缘出现较多的成骨细胞,并有骨组织形成;8周时钻孔区形成骨髓组织;2周时B组钻孔区有大量的成骨细胞,边缘有较多的骨组织形成。4周时钻孔区内充满新生骨小梁结构;8周时钻孔区内骨小梁趋于成熟。结论:髓芯减压和同种异体骨髓基质细胞移植,对兔股骨头缺血性坏死,有良好的治疗作用,且无明显地排异反应。     

Intravenous transplantation of allogeneic bone marrow mesenchymal stem cells and its directional migration to the necrotic femoral head

In this study, we investigated the feasibility and safety of intravenous transplantation of allogeneic bone marrow mesenchymal stem cells (MSCs) for femoral head repair, and observed the migration and distribution of MSCs in hosts. MSCs were labeled with green fluorescent protein (GFP) in vitro and injected into nude mice via vena caudalis, and the distribution of MSCs was dynamically monitored at 0, 6, 24, 48, 72 and 96 h after transplantation. Two weeks after the establishment of a rabbit model of femoral head necrosis, GFP labeled MSCs were injected into these rabbits via ear vein, immunological rejection and graft versus host disease were observed and necrotic and normal femoral heads, bone marrows, lungs, and livers were harvested at 2, 4 and 6 w after transplantation. The sections of these tissues were observed under fluorescent microscope. More than 70 % MSCs were successfully labeled with GFP at 72 h after labeling. MSCs were uniformly distributed in multiple organs and tissues including brain, lungs, heart, kidneys, intestine and bilateral hip joints of nude mice. In rabbits, at 6 w after intravenous transplantation, GFP labeled MSCs were noted in the lungs, liver, bone marrow and normal and necrotic femoral heads of rabbits, and the number of MSCs in bone marrow was higher than that in the, femoral head, liver and lungs. Furthermore, the number of MSCs peaked at 6 w after transplantation. Moreover, no immunological rejection and graft versus host disease were found after transplantation in rabbits. Our results revealed intravenously implanted MSCs could migrate into the femoral head of hosts, and especially migrate directionally and survive in the necrotic femoral heads. Thus, it is feasible and safe to treat femoral head necrosis by intravenous transplantation of allogeneic MSCs.     

A Canine Model of Femoral Head Osteonecrosis Induced by an Ethanol Injection Navigated by a Novel Template

There is no consensus on how to establish models of osteonecrosis of the femoral head (ONFH) in large mammals. The aim of this study was to investigate the effectiveness of a novel canine model of ONFH, induced by a navigated injection of absolute ethanol. Using three-dimensional reconstruction and rapid prototyping manufacturing techniques, a new template was designed and processed to navigate the ethanol injection. The femoral heads of 18 adult dogs were injected with ethanol. Macroscopic, X-ray and histological examinations were performed at 3, 6, and 9 weeks after the operation. Further, computed tomography (CT), magnetic resonance imaging (MRI), and radionuclide scans were performed 6 weeks postoperatively. Three weeks after the operation, the femoral heads showed evidence of osteonecrosis including increasing numbers of empty lacunae, decreased hematopoietic cells, and destroyed adipose tissue in the medullary cavity, which increased in severity at the subsequent follow-up evaluations at 6 and 9 weeks. Fractured trabeculae and fibrous tissue were noted 9 weeks postoperatively. Image analysis also revealed evidence of osteonecrosis, such as several osteopenic areas with sclerotic rims on the X-ray, several areas of low bone mineral density with sclerosis on the CT scan, increased uptake of the nuclide species in MRI, and an inhomogeneous long T2 signal on the radioisotopic images. Ethanol injection navigated by our novel template was successful in establishing a canine model of ONFH. This model can be used to test new treatment modalities for human ONFH.