Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.
The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold.
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.
大规模的骨干骨缺损是整形外科医生一个巨大的挑战。骨替代自体骨移植,目前认为是黄金标准治疗,是限量供应,并与收获有关的发病率有关。由于这些原因,组织工程骨构建体骨髓间充质干细胞相结合的骨传导支架已探索作为在整形外科手术自体移植的替代方法。
迄今为止,大多数的研究已经在临床相关的动物模型如狗,猪,羊1-3,但在原位,节段性,关键尺寸骨的小动物模型的缺陷,这些结构的初步评价执行(如只小鼠)可以有几个优点:(ⅰ)低费用,(ⅱ)可被操作大量的动物; (三)在对比大型动物模型中,小鼠品系的均匀限制在支架骨吸收一个个体差异第二骨形成和; (四)最重要的是,特异性抗体和基因靶向动物的可用性使参与骨愈合的生物学过程的评价。最后但并非最不重要的,使用小鼠的免疫缺陷的菌株还可以研究使用任一移植物或人来源的细胞,而不在小鼠的不良免疫反应。
尽管有上述优点,在小鼠体内大量骨干骨缺损的模型是稀疏。大多数这种模型的使用的骨固定用髓内钉,其填埋所述骨髓腔(从而限制材料的体积要测试),也通过不提供旋转和轴向稳定性2,4-7阻碍再现性。
当前研究的目的是(i)模仿临床骨不愈合的情况下,描述一个可重复的,关键的大小,分段,小鼠股骨缺损模型,这是精确和可重复的锁板osteosynth稳定ESIS,提供了高度稳定的生物力学环境8-10; (ⅱ)来说明具有两个潜在的骨代用品本模型和描述的骨形成分析可能被使用。
骨科相关的材料和小鼠装置的异位植入通常执行以评估骨形成各种支架13,14的能力。然而,重要的差异异位原位车型,其中包括原生骨信号因子,并与宿主骨形成细胞旁分泌的相互作用之间。
本研究建立了一个可重复的鼠大段,临界尺寸股骨缺损(3.5毫米,股骨长度约为20-25%)。考虑这种缺陷的大小和由所得接骨板提供的稳定性,该模型模仿临床上遇到萎缩骨不连。
在本研究中所选择的手术后的一段时间内,在与前述不愈合模型小鼠线,表示8〜12周4,9,15,16后缺乏足够的愈合。
最重要的是,reprodu无显著发病率和死亡率的1,2-与使用两个锁定板和执行截骨夹具的获得埚和稳定接骨,以及植入骨替代物的稳定性。这一结果也对比时,无论是外固定器或髓内钉用于4,5,17-24结果报告如下。对于外固定器的潜在缺点包括:变异刚度,销束的感染,松动销的,电势由于销与材料(4〜20的小鼠体重的百分比)的重量损伤。对于髓内钉潜在缺点包括:用指甲和关节面的医源性损伤髓腔的填充。
通过接骨板稳定其它鼠节段性,临界尺寸股骨缺损已与由毛刺和范围从1.5至2毫米的长度16,25创建骨缺损说明。在日E存在模式,利用夹具和埋弧焊丝的允许精确3.5毫米长的截骨不显著肌肉创伤。
然而,在执行一个应该考虑的问题几个关键点的过程成功:不要用小的小鼠(裸鼠或者重量低于25 g或未满8周),否则的铭牌应过长。当接近股骨,注意保护两个尾部坐骨神经和关节囊远端。适用于股骨的前侧的板,并且由于板的对准通过该第一螺钉的应用确定,照顾插入该第一螺钉时,平行板定位到股骨。
使截骨术前,照顾到骨干中部执行股骨的圆形解剖,以避免肌肉创伤。当执行截骨术,外科医生的助手必须抓紧指导和河畔GEON必须小心(i)不纠结锯丝,(ⅱ)使用导线的中间三分之二而施加恒定稳定张力,和(iii),以避免过量的运动,以获得直骨切口。
骨愈合是可能在本模型提供了一个骨移植物被使用。此外,该模型允许参与骨替换策略的机制时,无论是人起源移植物或细胞在良好标准化,大,节段性骨缺损用于进一步研究。
此外,在一行需要细化和减少使用在骨科相关的研究的动物目前的趋势,这种模式可在与体内成像技术,例如生物发光一起使用。这种非侵入性技术允许同时监测植入细胞的存活和组织愈合,而不需要动物牺牲26。
本模型的主要限制是两个承重条件和创建的,因为它们不完全模仿那些在人类临床上所遇到的骨缺损的体积。模型的其它局限性是:(i)该板的辐射不透明度,这可能需要移除所述板的离体的μCT分析之前,可能复杂的纵向放射检查结果的解释和,(ii)所述无力调节板刚度其中可能是骨形成27-30的关键力学参数。
必须牢记也,即使用骨同系移植或含矿物组分(具体碳酸钙)等的支架时,有些偏差都在微CT分析的分割过程中引入的,因为新形成的骨的密度部分地重叠无论是同基因移植密度或支架密度。由于这个原因,骨体积由微CT分析获得大多体现矿化组织(新形成的骨的体积加骨替代)11,26,31。
The authors have nothing to disclose.
作者要感谢瑞纳Bizios她对稿件宝贵意见。
α-MEM , Minimum Essential Medium Eagle | Sigma-Aldrich, France | M4526 | 500 ml |
Acropora sp. coral exoskeleton cubes, Biocoral® | Biocoral®, Inoteb, France | 3x3x3 mm cubes, autoclaving (121°C for 20 min) sterilization | |
Buprenorphine, Buprecare® | Axience, Pantin, France | 0.3 mg/ml | |
Xylazine, Rompun® 2% | Bayer HealthCare, Puteaux, France | 20 mg/ml | |
Ketamine, Ketamine 500® | Virbac, Carros, France | 50 mg/ml | |
Isoflurane, Forène® | Abbott, Arcueil, France | ||
Enrofloxacine, Baytril® 5% | Bayer HealthCare, Puteaux, France | 50 mg/ml | |
Pentobarbital, Dolethal® | Vétoquinol, Lure, France | 182,2 mg/ml | |
Anesthetizing box | Ugo Basile, Gemonio, Italy | 7900/10 | |
Plastic transparent sterile drape, BusterOpCover 30*45cm | Buster, Coveto, Montagu, France | 613867 | |
10% povidone iodine, Vétédine® Solution | Vétoquinol, Lure, France | 100 mg/ml | |
Titanium micro- locking plate, MouseFix Plate XL | RISystem AG, Davos, Switzerland, http://www.risystem.com/ | RIS.401.120 | 6 holes, 10 mm long and 1.5 mm wide, autoclaving (121°C for 20 min) sterilization or cold sterilzation (ethylene oxide) |
0.3 mm drill bit, Drill Bit 0.30 mm | RISystem AG, Davos, Switzerland, http://www.risystem.com/ | RIS.592.200 | autoclaving (121°C for 20 min) sterilization or cold sterilzation (ethylene oxide) |
Engine power | RISystem AG, Davos, Switzerland, http://www.risystem.com/ | AccuPen | Cold sterilzation (ethylene oxide) |
Screw driver, Handrill | RISystem AG, Davos, Switzerland, http://www.risystem.com/ | RIS.390.130 | autoclaving (121°C for 20 min) sterilization or cold sterilzation (ethylene oxide) |
Self-tapping locking screws, MouseFix Screw 2 mm | RISystem AG, Davos, Switzerland, http://www.risystem.com/ | RIS.401.100 | 2 mm long, 0.47 mm outer diameter and 0.34 mm core diameter, autoclaving (121°C for 20 min) sterilization or cold sterilzation (ethylene oxide) |
Jig,MouseFix XL Drill and Saw Guide | RISystem AG, Davos, Switzerland, http://www.risystem.com/ | RIS.301.103 | 3.5 mm between the slots, autoclaving (121°C for 20 min) sterilization or cold sterilzation (ethylene oxide) |
0.22-mm Gigli saws (0.22 mm Saws) | RISystem AG, Davos, Switzerland | ||
5.0 glycomer 631, Biosyn | Covidien, Vétoquinol, Lure, France | Tapper-cut needle | |
4.0 glycomer 631, Biosyn | Covidien, Vétoquinol, Lure, France | Tapper-cut needle | |
Xray, MX20 | Faxitron X-ray Corp, Edimex, Le Plessis Grammorie | ||
in vivo high-resolution microcomputed tomography, Skyscan 1176 | Skyscan, Aartselaar, Belgium | ||
Ex vivo high-resolution microcomputed tomography, Skyscan 1172 | Skyscan, Aartselaar, Belgium | ||
Resident software: Nrecon(v1.6.9)/Ctan(v.1.14.4) | Skyscan, Aartselaar, Belgium |