【作者】 张军；

【导师】 蔡锦方；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 一实验目的临床高能量损伤造成的开放性骨折常发生骨髓炎并骨质外露坏死,手术清创后常造成节段性骨缺损。感染性骨缺损的修复重建非常困难。我们试图通过模拟临床创伤后骨髓炎的发病机制建立动物模型,在此模型基础上以骨髓炎坏死皮质骨为研究对象,制备出部分脱钙骨基质并设法恢复其血运,同时结合显微外科技术来修复胫骨感染性骨缺损。二实验方法:(一)第一部分伴软组织缺损的创伤后骨髓炎皮质骨坏死动物模型的创建。以雄性Wistar大鼠为研究对象,随机分为两组,实验组和对照组各10只,共20只。在大鼠胫骨干制造4mm×8mm皮质骨缺损,将3×10~6cfu金黄色葡萄球菌和异物(纱布)接种到胫骨骨缺损处髓腔内,并将游离骨块回植到缺损区,外翻缝合软组织使骨质外露,对照组不接种金葡菌。术后2W、4W观察记录一般情况,行放射学、病理组织学检查。定量数据采用SPSS 12.0统计软件处理。术后通过病理组织学和放射学方法评价骨髓炎及骨坏死的情况。(二)第二部分大鼠骨髓炎坏死皮质骨脱钙骨基质制备及理化性质和生物力学测定。以大鼠骨髓炎坏死皮质骨和正常新鲜皮质骨作为研究对象,按改良Urist法将骨髓炎坏死皮质骨经脱钙脱脂处理得到部分脱钙骨基质,扫描电镜观察测量其孔隙度,全自动生化分析仪测量其钙含量并计算脱钙率,生物力学仪测量其形变—负荷曲线。(三)第三部分自体骨髓炎坏死皮质骨脱钙骨基质再血管化并修复骨缺损的实验研究。以骨髓炎骨坏死模型造模后的雄性Wistar大鼠为研究对象,随机分成两大组,腹部包埋组:其中实验组和对照组各9只,共18只;骨缺损修复组22只,共40只。所有大鼠取腹部切口显露腹壁浅动脉,将骨髓炎坏死皮质骨部分脱钙骨基质灭菌后,无菌条件下包埋于双侧腹壁浅动脉肌瓣中,对照组将未脱钙坏死皮质骨包埋于相同肌瓣中。腹部包埋组术后1W、2W、4W实验组和对照组各处死3只动物,取材行病理组织学检查观察血管化情况。骨缺损修复组22只大鼠于腹壁浅动脉肌瓣包埋后2W,将部分血管化的坏死皮质骨脱钙骨基质连同腹壁浅动脉带蒂肌瓣转移填充于胫骨骨髓炎骨缺损处,术后4W、8W、12W行经放射学和病理组织学检查骨髓炎及缺损修复情况。三结果(一)第一部分实验组大鼠术后体重下降,体温和WBC升高,放射学检查见髓腔内骨质破坏,骨皮质变薄,骨膜反应和软组织肿胀阴影,部分出现病理性骨折。病理组织学见髓腔内大量炎细胞浸润,骨小梁部分溶解、吸收、稀疏、坏死,骨细胞坏死,骨陷窝空虚。对照侧仅表现为轻微的炎症反应,无明显骨髓炎征象。(二)第二部分骨髓炎坏死皮质骨经脱钙脱脂后形成部分脱钙骨基质,平均钙含量为92.17±0.54 mg/g,脱钙率为38.1%。扫描电镜显示:坏死皮质骨脱钙骨基质表面凹凸不平,包含两种微孔结构,直径在7～15μm和0.1～0.4μm之间,微孔之间相互沟通共同形成筛孔样结构,与正常新鲜皮质骨DBM比较,无明显差异。生物力学测定:形变与负荷呈函数关系,随着负荷增加,形变逐渐增加,形变一负荷表现为一条平缓的上升曲线,与新鲜皮质骨形变-负荷曲线比较接近。(三)第三部分腹部包埋实验组1W时,部分脱钙骨基质内大量骨细胞坏死,骨陷窝空虚,周围肌肉组织中炎细胞浸润。2W时,哈弗氏管内偶见血管长入,主要集中在表层组织内,骨陷窝内仍空虚,无细胞或血管。4W时少许血管长入,仍以集中在表层为主,血管直径不等,分布不均,骨陷窝内可见成骨细胞,分布密度不均,无特征性骨结构。对照组2W,4W坏死皮质骨组织内均未见血管长入。骨缺损修复组术后4W,脱钙骨基质内出现了软骨化生,周围被纤维细胞包绕,靠近骨髓腔一侧可见炎细胞浸润。术后8W:部分脱钙骨基质大量软骨化,外层包被纤维组织和肌肉。术后12W:部分脱钙骨基质出现不同程度的软骨化和骨化,靠近干骺端一侧骨化明显,出现部分类似骨小梁的结构。放射学显示胫骨缺损修复区4W时,胫骨缺损清晰可见,移植组织密度低,无钙化征象。8W时,胫骨缺损仍清晰可见,移植组织密度低,无明显钙化征象,周围组织硬化,骨膜增生。12W时,胫骨缺损稍模糊,移植组织密度稍增高,与周围骨质有部分连接。四结论(一)第一部分大鼠胫骨造成皮质骨缺损并接种金葡菌和异物,骨块回植并骨外露,可以成功建立伴软组织缺损的创伤后骨髓炎皮质骨坏死动物模型。细菌、异物、创伤是建立此模型的必要条件,软组织外翻缝合骨外露更接近临床伴有软组织缺损的创伤后骨髓炎的实际情况。(二)第二部分创伤后骨髓炎坏死皮质骨经脱钙脱脂处理后可得到部分脱钙骨基质,既有三维的网状孔隙结构,又保持了一定的生物力学强度,既脱去部分钙质,暴露出骨基质中的细胞因子,又因源于自体组织不存在免疫排斥反应,具备了DBM的基本特性,并有独特的优点,具备再血管化可能和修复骨缺损的潜能。(三)第三部分自体骨髓炎坏死皮质骨部分脱钙骨基质包埋于自体腹壁浅动脉肌瓣中可以再血管化。部分血管化的坏死皮质骨脱钙骨基质连同腹壁浅动脉带蒂肌瓣转移填充于胫骨骨髓炎骨缺损,可以改善局部血运并修复骨髓炎骨缺损。更多还原

【Abstract】 一ObjectiveThe segmental bone defect often comes from debridement of necrosis cortical bone in post-trauma osteomyelitis.It is very difficult to repair the infect bone defect.We tried to create an animal model of post-trauma osteomyelitis according to clinical pathogenesy.Based on the animal model,the dead bone was prepared to be an partial decalcified bone matrix(DBM).Then we tried to revascularize the DBM to repair the infected tibia defect.二MethodsThe first part:Founding of animal model of necrosis cortical bone in post-trauma osteomyelitis with soft tissue defect.Staphylococcus aureus and foreign body were inoculated into the rat tibia medullary space through the bone defect.The bone block from the tibia defect was replanted onto the defect and was revealed by outpouching the soft tissue.Pathohistology and radiology were used to evaluated the osteomyelitis and bone necrosis.The second part:Preparation of decalcified bone matrix of necrosis cortical bone from the rat infect tibia and determination of the physico-chemical and vitodynamics.property.According to reforming Urist method,the partial decalcified bone matrix (DBM) was obtained from necrosis cortical bone of osteomyelitis by decalcification and defat.The porosity and calcium contents and deformation-lord curve were evaluated by scanning electron microscope and automatic biochemistry analyzer and vitodynamics machine created with of S.aureus and foreign body,while the bone was exposed in the air.It was evaluated with X-ray and pathology.After decalcified and defatted,the necrosis cortical bone was scanned by scanning electric-microscopy,measured the calcification by automatic biochemical machine,measured the biomechanics characteristic by biomechanics machine.The third part: The experimental study of autologous revascularized partial decalcified bone matrix to repair the osteomyelitis bone defect.The sterilized partial decalcified bone matrix was embedded into the superficial epigastric artery muscle flap to revascularize.Combined with the muscle flap vessel pedicle,it was transferred into the infected tibia defect.the revascularization and bone defect reparation,were observed by radiology and pathohistology 三ResultThe test tibia revealed feature of osteomyelitis.And the replanted bone was necrosis. The animal model of necrosis cortical bone in post-trauma osteomyelitis with soft tissue defect was founded.After decalcification and defat,the partial decalcification bone matrix was obtained from the necrosis cortical bone.Its calcium contents is 92.17±0.54 mg/g and decalcification rate 38.1%.Two kinds of cellular structure communicating each other were observed through scanning electron microscope with 7～15μm and0.1～0.4μm in diameters. Deformation and lord is functional relation.the deformation raised with the increased lord.Deformation-load curve was similar to the fresh cortical bone curve.Four weeks later, the necrosis cortical bone matrix embedded in the superficial epigastric artery muscle flap were revascularized and decalcified in the infected tibia defect during the later time.四ConclusionThe animal model of necrosis cortical bone in post-trauma osteomyelitis with soft tissue defect was successfully created in our method.After decalcification and defat,the necrosis cortical bone revealed porous structure and vitodynamics intensity.The necrosis cortical bone matrix embedded in the muscle flap was able to revascularize and repair the infected bone defect.更多还原