建立小鼠骨干股骨骨折模型
1Postgraduate Program in Biological Sciences – Biophysics, Institute of Biophysics Carlos Chagas Filho,Federal University of Rio de Janeiro, 2Postgraduate Program in Surgical Sciences, Faculty of Medicine,Federal University of Rio de Janeiro, 3Institute of Biomedical Sciences,Federal University of Rio de Janeiro
Summary
该协议描述了一种外科手术，用于在小鼠股骨中建立骨干骨折，该骨折用髓内线稳定，用于骨折愈合研究。
Abstract
骨骼具有显着的再生能力。然而，骨折愈合是一个复杂的过程，根据病变的严重程度以及患者的年龄和整体健康状况，可能会发生失败，导致愈合延迟或不愈合。由于高能创伤和衰老导致的骨折数量不断增加，迫切需要开发基于骨骼/间充质干细胞/基质细胞和仿生生物材料相结合的创新治疗策略来改善骨修复。为此，使用可靠的动物模型对于更好地了解决定愈合结果的关键细胞和分子机制至关重要。在所有模型中，小鼠是首选的研究模型，因为它提供了多种用于实验分析的转基因菌株和试剂。然而，由于小鼠体型小，在小鼠中建立骨折可能在技术上具有挑战性。因此，本文旨在展示通过软骨愈伤组织形成的小鼠骨干股骨骨折的手术建立程序，该骨折由髓内线稳定，类似于最常见的骨修复过程。
Introduction
骨骼是一个重要且功能多样的器官。骨骼的骨骼使身体姿势和运动成为可能，保护内脏器官，产生整合生理反应的激素，并且是造血和矿物质储存的场所1.如果骨折，骨骼具有非凡的再生能力，可以完全恢复其受伤前的形态和功能。愈合过程始于血肿和炎症反应的形成，这诱导骨膜、骨内膜和骨髓的骨骼干/祖细胞的活化和凝聚，以及随后的分化形成软骨愈伤组织。然后，断裂末端的桥接通过类似于软骨内骨形成的过程发生，其中软骨支架膨胀然后矿化，形成坚硬的骨性愈伤组织。最后，硬愈伤组织逐渐由破骨细胞和成骨细胞重塑，以恢复原始骨骼结构 2,3。
尽管骨折愈合过程相当稳健，但它涉及一系列错综复杂的事件，并受到几个个体因素的显着影响，包括患者的一般健康状况、年龄和性别，以及损伤因素，例如骨折的机械稳定方式、感染的发生以及周围软组织损伤的严重程度4，5,6.因此，失败很常见，导致骨不连的发展，这极大地影响了患者的康复和生活质量 7,8。由于高能量创伤和衰老导致的骨折数量增加，以及治疗费用高昂，不愈合骨折已成为全世界卫生系统的负担 9,10。这种日益增加的负担凸显了对创新治疗策略的迫切需要，以基于骨骼/间充质干细胞/基质细胞和仿生生物材料的组合来改善骨修复11,12 13,14。
为了实现这一目标，动物模型已被广泛用于旨在了解骨折愈合机制基础生物学的研究，以及旨在设计新的治疗策略以促进骨修复的概念验证临床前研究15,16,17。小动物模型，如小鼠，非常适合骨折愈合研究，因为转基因菌株和试剂可用于实验分析，而且维护成本低。此外，小鼠具有快速的愈合时间过程，这允许对修复过程的所有阶段进行时间分析15。然而，动物的小体型可能会给骨折的手术产生带来挑战，其固定模式类似于人类的固定模式。该协议描述了一种简单且低成本的小鼠骨折愈合模型，使用用髓内线稳定的开放式股骨截骨术，类似于最常见的骨修复过程，通过软骨痂的形成，并且可用于需要进入骨折部位的基础和转化研究。
Protocol
所有实验均已获得里约热内卢联邦大学健康科学中心动物使用和护理委员会的批准（协议编号101/21）。本研究使用10-12周龄（25-30g体重）的雄性Balb/c小鼠。每只小鼠的手术过程大约需要 15-20 分钟。在每次手术之前，所需的器械（列在 材料表中）必须组织在覆盖手术台的无菌手术区域（图1A）。金属手术器械必须在123°C的自密封信封中高压灭菌30分钟。一次性物品…
Representative Results
评估外科手术在骨折方面是否成功的最简单和最直接的方法是 X 射线成像。可以在手术后立即进行 X 光片，小鼠仍处于麻醉状态，随后在骨折后 7 天、14 天和 21 天进行 X 光检查，以评估愈伤组织形成和进展。可接受的骨折模式是皮质完全破裂，钢丝正确放置在髓管内，并且骨折线是横向的（与骨轴成 90° 角）、斜的（弯曲或倾斜的模式，没有碎片位移）或短斜（相对于骨轴约 30°）（<strong class="x…
Discussion
随着全球骨折数量的增加 9,10,25，愈合不愈合的创新治疗方法变得越来越紧迫。由于骨折愈合涉及对在很长一段时间内发生的事件的复杂而紧密的总结3，因此使用有效的动物模型对于提高我们对决定骨修复成功与否的机制的理解以及选择有效的药物和治疗方案至关重要16,17<sup class="xr…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作由里约热内卢州卡洛斯·查加斯·菲略研究支持基金会（FAPERJ）资助。
Materials
| Alcohol 70º | Merck | 109-56-8 | Or any general available supplier |
| Canada balsam (mounting medium) | Merck | C1795 | Or any general available supplier |
| Cefazoline | ABL | Not applicable | Similar brands of the item may be used according to local availability |
| Coverslip | Merck | CSL284525 | Or any general available supplier |
| Dental X-Ray Generator | Focus | – | Sold by Instrumentarium Dental Inc. |
| DEPC water | Merck | W4502 | Or any general available supplier |
| Dissecting Scissor | ABC Instrumentos | 0327 | Similar brands of the item may be used according to local availability |
| EDTA | Vetec | 60REAVET014340 | Similar brands of the item may be used according to local availability |
| Eosin solution | Laborclin | EA-65 | Similar brands of the item may be used according to local availability |
| Ethanol P.A | Vetec | 60REAVET012053 | Similar brands of the item may be used according to local availability |
| Gauze pads | Cremer | Not applicable | Or any general available supplier |
| Harris Hematoxylin Solution | Laborclin | 620503 | Similar brands of the item may be used according to local availability |
| Heating pad | Tonkey Electrical Technology | E114273 | Similar brands of the item may be used according to local availability |
| Histological slides | Merck | CSL294875X25 | Or any general available supplier |
| Histology cassettes | Merck | H0542-1CS | Or any general available supplier |
| Hydrochloric acid – 37% | Merck | 258148 | Similar brands of the item may be used according to local availability |
| Insulin syringe | BD | 324918 | Or any general available supplier |
| Iodopovidone sponge | Rioquímica | 372106 | Or any general available supplier |
| Ketamine hydrochloride | Ceva | Not applicable | Similar brands of the item may be used according to local availability |
| Lacribel collyrium | Cristalia | Not applicable | Similar brands of the item may be used according to local availability |
| Microtome | Leica | 149AUTO00C1 | |
| Mouse Tooth Forceps Tweezer | ABC Instrumentos | 0164 | Similar brands of the item may be used according to local availability |
| Needle 26 G | BD | 2239 | Or any general available supplier |
| Needle Holder | Golgran | 135-18 | Similar brands of the item may be used according to local availability |
| Nonresorbable Nylon Suture thread nº 6 | Atramat | C1546-NT | Or any general available supplier |
| Paraffin | Exodo | 8002 – 74 – 2 | Similar brands of the item may be used according to local availability |
| Paraformaldehyde | Sigma | 30525-89-4 | Similar brands of the item may be used according to local availability |
| PBS 1x | Lonza | BE17-516F | Similar brands of the item may be used according to local availability |
| Resorbable Nylon Suture thread nº 6 | Atramat | C1596-45B | Or any general available supplier |
| Rod Wire SS CrNi 0.016" | Orthometric | 56.50.2016 | |
| Scalpel nº 11 | Descarpak | 15782 | Or any general available supplier |
| Serrated Tip Tweezer | Quinelato | QC.404.12 | Similar brands of the item may be used according to local availability |
| Shaver | Phillips | Not applicable | Similar brands of the item may be used according to local availability |
| Surgical tape | 3M | 2734 | Or any general available supplier |
| Surgical tnt field | Polarfix | 6153 | Or any general available supplier |
| Tramadol hydrochloride | Teuto | Not applicable | Similar brands of the item may be used according to local availability |
| Water bath for histology | Leica | HI1210 | |
| Xylazine hydrochloride | Ceva | Not applicable | Similar brands of the item may be used according to local availability |
| Xylene | Dinamica | 60READIN001105 | Similar brands of the item may be used according to local availability |
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Citar este artigo
Braga Frade, B., Dias da Cunha Muller, L., Bonfim, D. C. Establishing a Diaphyseal Femur Fracture Model in Mice. J. Vis. Exp. (190), e64766, doi:10.3791/64766 (2022).