这项工作得到了国防部 (DoD) 美国陆军医学研究和装备司令部 (USAMRMC) 国会定向医学研究项目 (CDMRP) (PR121604) 和国家关节炎和肌肉骨骼研究所赠款的支持。和皮肤病 (结缔组织国立研究院), NIH R01 AR068332 桑卡尔。

以下程序是由印第安纳大学医学院的动物保育和使用委员会 (IACUC) 批准的。所有的生存手术都是在无菌条件下进行的, 如 NIH 指南所述。疼痛和感染的风险管理与适当的止痛药和抗生素, 以确保成功的结果。
1. 麻醉和准备
<ol><li>用腹腔 (ip) 路线对老鼠进行称量, 并麻醉氯胺酮 (100 毫克/千克) 和甲苯噻嗪 (10 毫克/千克) 的混合物。将鼠标放在一个空笼子里并监视它, 直到它完全镇静下来。</li>
	<li>使用脚趾捏反射确保鼠标镇静。将眼膏涂抹在眼睛上, 防止其干燥。</li>
	<li>从右肢上取出皮毛。用碘基擦洗和70% 乙醇擦拭手术部位。从膝盖中心开始擦洗手术部位, 并向外进行环形清扫。重复这3x 与新鲜的磨砂, 结束70% 乙醇。</li>
	<li>术前剂量盐酸丁丙诺啡镇痛 (0.03 毫克/千克) 皮下即刻疼痛管理。</li>
	<li>将鼠标放在无菌手术垫覆盖的加热垫上。</li>
</ol>2. 手术方法
注: 骨折前, 应在手术前对小鼠的特定应变、年龄和性别进行经验性测定。这种手术方法是为 C57BJ6 雄性小鼠在10周的年龄进行优化。
<ol><li>将鼠标放在背部, 弯曲手术腿的膝盖。使用手术刀刀片, 使一个1.5 厘米的切口集中在膝关节。</li>
	<li>用镊子将髌骨侧向移位以暴露股骨远端。在滑车槽的中心插入一个1.5 长25口径不锈钢皮下注射针, 沿着髓质管的长度逆行, 并通过股骨的近端。采取 X 射线, 以确保正确放置 pin。 注: 针应从鼠标的背侧离开以创建导丝的路径。</li>
	<li>通过针的轴, 通过4长36口径的钨导丝, 进入中心的股骨远端和退出的斜角在鼠标的背侧。</li>
	<li>在成功放置导丝后, 小心地取出25口径的针, 轻轻地拉在轮毂上, 同时保持四肢和导丝到位。通过 x 射线确认导丝的位置。</li>
	<li>从撞击盘上方34.6 厘米的高度保持391克重量 (图 1A)。将股骨横放在两个支撑点上, 使股骨的股骨粗隆间和髁上区域在支撑铁砧 (图 1B) 和肢体的侧边上, 面对加载点 (图1C).放下重量, 并小心地从设备上取出鼠标后立即断开。</li>
	<li>用 X 射线确认骨折位置。</li>
	<li>在导丝上插入24口径不锈钢皮下导管, 以稳定股骨骨折。 注: 此应用程序可能需要一些力, 因为进入点是使用一个较小的直径针产生。这种直径的差异有效地防止了24尺杆通过股骨近端的潜在迁移。插入深度可以手动感觉, 因为钝管满足较大的转子皮质骨。</li>
	<li>在去除导丝前, 用 X 射线确定钢棒的位置和股骨骨折的稳定性。</li>
	<li>用钢丝钳切开股骨远端的多余油管。用镊子将暴露的油管埋在髁表面下, 用钳子轻轻向下用力, 小心不要使膝关节脱臼。</li>
	<li>用镊子重新定位髌骨。用5-0 可吸收缝线关闭切口部位。</li>
</ol>3. 术后管理
<ol><li>手术后, 小鼠可通过ip 路线注射多达500µL 的无菌生理盐水, 以帮助他们在术后恢复。</li>
	<li>在加热的恢复床上监视动物, 直到他们从手术中醒来。一旦走动, 把他们送回笼子里去。</li>
	<li>在手术后的几天里, 继续密切监视老鼠, 以确保他们恢复正常, 恢复机动性。管理盐酸丁丙诺啡镇痛 (0.03 毫克/千克) 皮下每6小时皮下手术后3天, 并根据需要此后。避免使用非甾体抗炎药物, 因为它们已经被证明会损害手术后的骨愈合。</li>
</ol>

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这篇手稿的作者没有什么可透露的。作者进一步指出, 对本手稿所报告的研究中使用的所有材料的完全访问没有任何限制。

本手术的目的是在小鼠中产生标准的闭合性股骨骨折。该模型的一个主要优点是, 内固定发生在骨折产生后, 从而避免了髓内棒的角度。也许这个协议最关键的方面是在股骨中段形成一个标准化的横向骨折, 因为骨折几何取决于施加的弯曲力和后肢的位置。在弯矩时, 股骨的定位不当会导致偏斜或粉碎性骨折。重量和下落高度必须是经验主义地预定的, 因为他们取决于老鼠的年龄、性和劳损。采用装...

骨折是肌肉骨骼系统中最常见的损伤之一, 与巨大的社会经济负担有关, 包括预计在美国每年超过250亿美元的治疗费用1, 2。虽然大多数骨折愈合无事故, 愈合与大量停机和生产力损失。大约 5-10% 的骨折导致延迟愈合或不愈合, 由于年龄或其他基本的慢性健康状况, 如骨质疏松症和糖尿病3,4,5。目前尚无 FDA 批准的药理治疗, 可促进有效的骨愈合, 缩短恢复时间。
骨折愈合是一个复杂和高度动态的过程, 涉及多种细胞类型的协调。因此, 全面了解与骨再生相关的细胞和分子事件, 对于确定加速这一过程的治疗目标至关重要。与其他人类疾病一样, 建立一个高度顺从和可再生的动物模型是研究骨愈合的关键。较大的动物, 如绵羊和猪, 有骨重塑的性质和生物力学类似于人类, 但昂贵, 需要大量的愈合时间, 并不容易服从基因操纵6。另一方面, 小动物模型, 如大鼠和小鼠, 提供了许多好处, 包括易于处理, 维护成本低, 繁殖周期短, 愈合时间缩短7。此外, 鼠标基因组是完全有序的, 允许快速操作和生成的遗传变异。因此, 鼠标是一个强大的模型系统, 研究人类疾病, 伤害和修复8。在人类中, 像骨质疏松症和糖尿病这样的并发症增加了延迟愈合的可能性。现有的一些小鼠模型可以用来研究骨质疏松症和糖尿病并发症对骨损伤和愈合的影响。患有骨质疏松症的患者在骨折愈合后的晚期骨形成明显减少9。去卵巢 (OVX) 小鼠表现出快速骨丢失和延迟骨愈合类似于绝经后骨质疏松症10,11。此外, 许多类型的 I 型和 II 型糖尿病的小鼠模型模仿的低骨质量表表和受损骨折愈合在人类看到11。此外, 小鼠骨折模型是研究愈伤组织复杂生物过程的多功能平台, 探索加速骨组织再生的新的治疗策略。
尽管骨结构和新陈代谢有差异, 但在小鼠和人类中, 骨折愈合的整个过程仍然非常相似, 包括软骨和膜内骨化的结合, 然后是骨重塑。软骨骨化包括招募祖细胞到较不机械稳定的区域周围的骨折间隙, 他们分化成软骨细胞, 肥大和矿化软骨产生柔软的愈伤组织。第二波祖细胞浸润愈伤组织, 分化为成熟的成骨细胞, 分泌新的骨基质12,13,14,15。在膜内骨化过程中, 骨膜和骨内表面的祖细胞直接分化为基质, 从而促进骨折间隙9、11、12 的衔接. ,13。在一起, 软骨和膜内血肿骨化相接导致了坚硬愈伤组织的发展, 随着时间的推移进一步改建, 形成一个强大的辅助骨, 能够支持机械负荷13,14 ,15。在健康的人, 愈合过程需要大约3月, 与仅35天在老鼠16。
骨折愈合通常被研究使用开放或闭合的外科模型17。开放性手术方法, 如产生严重的缺陷或完整的截骨, 规范损伤位置和几何, 以减少粉碎性骨折造成的偏差。截骨术作为一个优秀的模型来研究不愈合背后的基础机制, 因为愈合往往延迟比较闭合性骨折。此外, 需要硬外固定, 以稳定 osteotomized 骨, 这意味着再生将主要依赖于膜内骨化。开放式手术方法使用的设备, 如锁定钉, 别针夹子, 锁定板, 以提供轴向和旋转稳定性的骨折肢体;然而, 这些设备是昂贵的, 需要大量的时间在手术18,19,20,21。另一方面, 闭合模型是稳定的一个简单的髓内固定装置, 允许足够的不稳定, 以刺激软骨愈合。因此, 闭合断裂模型不容易模仿非联合的条件。内固定技术, 如髓内针, 指甲, 和加压螺钉, 是有利的, 因为他们便宜, 易于使用, 并尽量减少手术时间21,22,23。在某些情况下, 在骨折前插入髓内针, 但是髓内针的弯曲会导致股骨骨折的角或移位, 从而造成变的愈伤组织大小和愈合。在闭合模型中, 断裂位置和几何更难标准化, 因为它们是使用三点弯曲装置产生的, 其中骨干的重量被丢弃。然而, 用适当的技术, 这种手术方法提供了快速和一致的结果。此外, 闭合性骨折模型作为临床相关工具, 用于研究由高强度撞击或机械应力引起的骨折22。
这项手术协议是根据以前描述的方法, 使用髓内针稳定骨折股骨大鼠和小鼠22,24,25。首先, 通过髁间凹槽插入一个小直径的髓内针, 以建立一个入口点, 并在使用重力依赖性三点的股骨中段上产生横向骨折之前引入导丝。弯曲装置。在成功地产生闭合性股骨骨折后, 将一条直径较大的髓内棒结合在导丝上, 以稳定股骨骨折。这种方法避免了骨折时髓内针角引起的延迟愈合的风险, 因为在骨折后放置杆后, 可以重新定位和优化股骨损伤的稳定性。

<table><tbody><tr><td>Oster Minimax Trimmer</td><td>Animal World Network</td><td>78049-100</td><td></td></tr><tr><td>POVIDONE-IODINE</td><td>Thermo Fisher Scientific</td><td>395516</td><td></td></tr><tr><td>OPHTHALMIC OINTMENT</td><td>Thermo Fisher Scientific</td><td>NC0490117</td><td></td></tr><tr><td>Styker T/Pump Warm Water Recirculator</td><td>Kent Scientific Corporation</td><td>TP-700</td><td></td></tr><tr><td>1ml Sub-Q Syringe</td><td>Thermo Fisher Scientific</td><td>309597</td><td></td></tr><tr><td>ENCORE Sensi-Touch PF</td><td>Moore Medical LLC</td><td>30347</td><td>Latex, powder-free surgical glove</td></tr><tr><td>PrecisionGlide 25G Hypodermic Needles</td><td>Thermo Fisher Scientific</td><td>14-826-49</td><td></td></tr><tr><td>Ultra-High-Temperature Tungsten Wire,</td><td>McMaster-Carr</td><td>3775K37</td><td>0.005&quot; Diameter, 1/16 lb. Spool, 380&#039; Long</td></tr><tr><td>304 stainless steel, 24G thin walled tubing</td><td>Microgroup Inc</td><td>304h24tw-5ft</td><td></td></tr><tr><td>#15 Scalpel Blades</td><td>Fine Science Tools</td><td>10015-00</td><td></td></tr><tr><td>#10 Scalpel Blades</td><td>Fine Science Tools</td><td>10010-00</td><td></td></tr><tr><td>Narrow Pattern Forceps</td><td>Fine Science Tools</td><td>11002-12</td><td>Serrated/Straight/12cm</td></tr><tr><td>Iris Forceps</td><td>Fine Science Tools</td><td>11066-07</td><td>1x2 Teeth/Straight/7cm</td></tr><tr><td>Dissector Scissors</td><td>Fine Science Tools</td><td>14081-09</td><td>Slim Blades/Angled to Side/Sharp-Sharp/10cm</td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>14058-11</td><td>ToughCut/Straight/Sharp-Sharp/11.5cm</td></tr><tr><td>Olsen-Hegar Needle Holder with Suture Cutter</td><td>Fine Science Tools</td><td>12002-12</td><td>Straight/Serrated/12cm/with Lock</td></tr><tr><td>Crile Hemostat</td><td>Fine Science Tools</td><td>13004-14</td><td>Serrated/Straight/14cm</td></tr><tr><td>Tungsten Wire Cutter</td><td>ACE Surgical Supply Co., Inc.</td><td>08-051-90</td><td>ACE #150 Wire Cutter, tungsten carbide tips</td></tr><tr><td>3-0 VICRYL Suture</td><td>Ethicon Suture</td><td>J423H</td><td>3-0 VICRYL UNDYED 27&quot; FS-2 CUTTING</td></tr><tr><td>piXarray 100 Digital Specimen Radiography System</td><td>Bioptics, Inc</td><td></td><td>Cabinet x-ray system</td></tr><tr><td>Einhorn 3-Point Bending Device</td><td>N/A</td><td>N/A</td><td>Custom Built</td></tr></tbody></table>

the generation of closed femoral fractures in mice: a model to study bone healing

骨骨折给病人带来了巨大的社会经济负担, 除了严重影响他们的生活质量。促进有效骨愈合的治疗策略是不存在和高需求的。为了了解与骨再生相关的复杂生物过程, 需要有效和可再生的骨折愈合动物模型。许多动物模型的骨折愈合已经产生了多年;然而, 小鼠骨折模型最近成为研究骨愈合的有力工具。各种开放和封闭的模型已经开发, 但闭合性股骨骨折模型突出作为一个简单的方法, 以产生快速和重现性的结果, 在生理上相关的方式。该手术方案的目的是在小鼠中产生单边闭合性股骨骨折, 并通过插入髓内钢棒促进股骨骨折后稳定。虽然诸如钉子或螺钉等设备提供了更大的轴向和旋转稳定性, 但使用髓内棒可以为一致愈合结果提供足够的稳定, 而不会产生新的骨组织缺陷或损害附近的软组织。影像学是用来监测愈伤组织的形成, 骨愈合, 并随后重塑骨痂的进展。骨愈合的结果通常与愈合的骨强度和测量的扭转试验。然而, 了解骨折修复相关的早期细胞和分子事件是研究骨组织再生的关键。髓内固定小鼠闭合性股骨骨折模型是研究骨折愈合的一个有吸引力的平台, 并对加速愈合的治疗策略进行评价。

通过影像学检查, 对手术过程的成功实施进行了监测。关键步骤包括插入髓内针, 放置导丝, 在股骨中段诱导横断, 并与髓内杆适当稳定 (图 2Ai - 2Aiv)。在手术后28天内用每周影像影像监测骨折愈伤组织的愈合进展 (图 2B)。10-16 天骨折后, 软骨细胞发生肥厚, 产生矿化软骨, 形成突出的柔软愈伤组织。
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Watch this Scientific Journal Video about The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing at JoVE.com

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

小鼠闭合性股骨骨折模型是研究骨折愈合的有力平台, 是加速骨再生的新的治疗策略。这项手术的目的是在小鼠中产生单侧闭合性股骨骨折, 用髓内钢棒稳定股骨。

小鼠闭合性股骨骨折的形成: 骨愈合模型的研究

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

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Research

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JoVE Core

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Anatomy and Physiology

Medicine

Unit 1

Bone Tissue and the Skeletal System

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15.1K 次观看.  Indiana University School of Medicine. 小鼠闭合性股骨骨折模型是研究骨折愈合的有力平台, 是加速骨再生的新的治疗策略。这项手术的目的是在小鼠中产生单侧闭合性股骨骨折, 用髓内钢棒稳定股骨。

视频: The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Pseudofracture: An Acute Peripheral Tissue Trauma Model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.1-9 Many studies have begun to assess these changes in the reactivity of the immune system following trauma.10-15 
Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.16-21
The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.22-30
This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.
The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.
Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. 31
Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. 32-36 The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. 37

Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.

Pseudofracture:急性周围组织的创伤模型

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; ...

科研

医学

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

一个简单的临界尺寸股骨缺损模型小鼠

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

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.

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.

在小鼠节段性股临界尺寸缺陷模型的建立接骨板稳定

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 ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

髓内锁钉股骨截骨标准化固定用于分析正常和有缺陷的骨愈合小鼠

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

骨科手术的小鼠模型研究术后认知功能障碍与组织再生

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

标准化生物力学条件下小鼠软骨骨折愈合的微创模型分析

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing therapeutics and devices. However, available injury models are plagued by inconsistency resulting in wasted animals and resources and imperfect data. To address this problem of fracture heterogeneity, the purpose of the method described herein is to optimize fracture generation parameters specific to each animal and yield a consistent fracture location and pattern. This protocol accounts for variations in bone size and morphology that may exist between mouse strains and can be adapted to generate consistent fractures in other species, such as rat. Additionally, a cost-effective, adjustable fracture apparatus is described. Compared to current stabilized fracture techniques, the optimization protocol and new fracture apparatus demonstrate increased consistency in stabilized fracture patterns and locations. Using optimized parameters specific to the sample type, the described protocol increases the precision of induced traumas, minimizing the fracture heterogeneity typically observed in closed-fracture generation procedures.

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

鼠类闭合稳定骨折的断裂装置设计与协议优化

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

开发Wistar白化大鼠股骨截骨模型的协议

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

带稳定针的小鼠股骨横断

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

生物学

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

建立小鼠骨干股骨骨折模型

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...