一种可靠、可重复的可重复的大鼠部分股骨缺陷模型
Summary
在体内, 哺乳动物的临界骨缺损模型是必不可少的研究愈合机制和骨科治疗。在这里, 我们介绍了一种方案, 以创建可重复的, 节段, 股骨缺损在大鼠稳定使用外固定。
Abstract
骨科研究在很大程度上依赖于动物模型来研究骨愈合的机制在体内, 以及研究新的治疗技术。临界大小的节段缺陷是具有挑战性的治疗临床, 研究工作可以受益于一个可靠的, 动态的小动物模型的节段股骨缺陷。在本研究中, 我们提出了一个优化的手术方案, 以一致和重现5毫米临界骨干缺损在大鼠股骨稳定与外部固定器。采用定制夹具对4根基什内尔电线进行了骨固定器切除术, 并采用合适的外固定装置进行了稳定。一个振荡骨锯被用来制造缺陷。单独植入胶原蛋白海绵或浸泡在 rhBMP-2 中的胶原蛋白海绵, 并使用 x 光片在12周内监测骨愈合情况。12周后, 对大鼠进行了牺牲, 并对切除和治疗股骨进行了组织学分析。骨缺损只含有胶原蛋白海绵导致不愈合, 而 Rbmp-2 治疗导致骨膜粘液和新的骨重塑形成。植入后动物恢复良好, 外固定成功地稳定了股骨缺损, 持续12周。这种简化的手术模式可以很容易地应用于研究骨愈合和测试新的骨科生物材料和再生疗法在体内。
Introduction
骨科创伤手术的重点是治疗广泛的复杂骨折。由于周围肌肉和骨膜再生能力下降以及局部血管生成失败, 临床上证明很难治疗严重的过敏性骨缺损.现代治疗技术包括骨移植、延迟骨 (masquelet) 的手术固定、骨移植、骨移植、融合或截肢2,3,4。在大多数患者谁具有流动功能保存后, 他们的创伤, 功能良好的远端肢体, 肢体救助显然是一个更好的治疗方案5。这些抢救治疗往往需要在一个漫长的疗程分阶段的手术干预。一些作者认为, 外固定优于内固定为这些应用相比, 由于减少组织损伤在体内植入表面积, 并增加术后可调性固定器6。然而, 一项前瞻性的随机对照试验目前正在进行中, 以帮助澄清这种争议的内固定对外外固定严重开放骨折的胫骨7。不幸的是, 无论选择哪种治疗方法, 显著的并发症和故障率持续 8,9。对于节段性骨丢失, 外科医生必须面对构成重大挑战的节段性过敏性缺陷。节段缺损的矫正必须最大限度地稳定骨骼, 同时增强成骨过程10,11。
由于关键大小的节段缺陷的临床重要性, 但体积较低, 有效、可重复的动物模型是必要的, 以使研究团队能够推进治疗技术并最终改善临床结果。研究人员需要研究哺乳动物动物模型中的体内生理愈合机制。虽然这种外固定模型已经存在12、13、14、15, 但我们希望为未经治疗的动物提供更可靠的不结合方法, 通过选择价格合理的固定器材料, 并概述了一个简单的手术协议, 便于应用于未来的研究。该方案的主要目的是建立一个可靠和可重复的模型, 为大鼠的关键过敏性缺损。通过评估大鼠股骨在12周内的稳定性和骨愈合情况, 对该手术进行了评估。次要目标包括: 使负担得起的模式尽可能具有成本效益, 简化手术方法和稳定, 并确保对动物的道德关怀。作者和研究小组对一系列不同的生物材料和潜在的再生疗法进行了初步实验, 以改善这种节段缺陷的愈合。
Protocol
本研究中使用的老鼠根据《动物安乐死 AVMA 准则: 2013年第16版》接受日常护理。威斯康星大学麦迪逊分校动物护理和使用机构委员会在项目开始前对这一实验方案进行了评估并批准。 1. 动物 使用超繁殖的 Sprague-Dawley 雄性大鼠, 重约350克。 2. 骨形态发生蛋白-2 (Rhmbp-2) 浸渍海绵支架的制备 注: 支架的准备工?…
Representative Results
在一名助理的帮助下, 一名外科医生在大约一小时内进行了手术。手术优化后, 术中和术后并发症大大减少, 使用夹具装置可确保大小一致 (5 x 3 x 3 mm) 和股骨缺损定位。从麻醉中恢复后, 大鼠立即处于流动状态, 似乎没有任何改变的行为模式;他们的步态并不像过敏, 他们似乎也没有被外部固定器打扰。 选择非螺纹 k 线的最近端引…
Discussion
骨科损伤的小动物模型, 如完整的骨折, 使研究, 探索成骨的机制和评估生物材料的治疗潜力20。本研究引入了由自定义外部固定器稳定的大鼠节段缺陷模型, 实验室和生物医学工程团队可以很容易地复制该模型, 以进一步研究承重骨合成骨修复。
此前在大鼠模型中使用临界大小缺陷的研究通常依赖于 21、22、</sup…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院设备赠款1S10OD023666-01 的支持, 并通过威斯康星大学骨科和康复系以及医学和公共卫生学院提供了额外的支持。我们要感谢 UW 的 Carone 癌症中心支助赠款 P30 CA014520 和使用他们的小动物成像设施, 以及国家卫生研究院培训赠款 5T35OD011078-08 h. martin 的支持。我们还感谢迈克尔和玛丽·苏·香农对肌肉骨骼再生伙伴关系的支持。
Materials
| 0.9% Sterile Saline | Baxter | 2F7124 | Used for irrigating wound and rehydration |
| 10% Iodine/Povidone | Carefusion | 1215016 | Used to prep skin |
| 10% Neutral Buffered Formalin | VWR | 89370094 | Used as fixative |
| 1mm non-threaded kirschner wire | DePuy Synthes | VW1003.15 | Sterilized, used for the most proximal pin |
| 1mm threaded kirschner wire | DePuy Synthes | VW1005.15 | Sterilized, used for the 3 most distal pin slots |
| 2×2 gauze | Covidien | 4006130 | Sterilized, used to prep skin and absorb blood |
| 4-0 Vicryl Suture | Ethicon | 4015304 | Used to close muscle and skin layers |
| 4-40 x 0.25",18-8 stainless steel button head cap screws | Generic | External fixator assembly | |
| 4200 Cordless Driver | Stryker | OR-S-4200 | Used to drill kirschner wires |
| 4×4 gauze | Covidien | 1219158 | Sterilized, used to absorb blood |
| 70 % Ethanol | Used to prep skin | ||
| Baytril | Bayer Healthcare LLC, Animal health division | 312.10010.3 | Added to water as an antibiotic |
| Cefazolin | Hikma Pharmaceuticals | 8917156 | Pre-op antibiotic |
| CleanCap Gaussia Luciferase mRNA (5moU) | TriLink Biotechnologies | L-7205 | Modified mRNA encoding for Gaussia Luciferase, keep on ice during use |
| Coelenterazine native | NanoLight Technology | 303 | Substrate for Guassia Luciferase, used to assess luciferase activity in vivo |
| Double antibiotic ointment | Johnson & Johnson consumer Inc | 8975432 | Applied to pin sites post-op as wound care |
| Dual Cut Microblade | Stryker | 5400-003-410 | Used to create 5mm defect in femur |
| Ethylenediamine Tetraacetic Acid (EDTA) | Fisher | BP120-500 | Used to decalcify bone to prep for histology |
| Extended Release Buprenorphine | ZooPharm | Used as 3 day pain relief | |
| Fenestrated drapes | 3M | 1204025 | Used to establish sterile field |
| Handpiece cord for TPS | Stryker | OR-S-5100-4N | Used to create 5mm defect in femur |
| Heating pad | K&H Pet Products | 121239 | Rat body temperature maintenance |
| Hexagonal head screwdriver | Wiha | 263/1/16 " X 50 | External fixator tightening |
| Induction chamber | Generic | Anesthesia for rats | |
| Infuse collagen sponge with recombinant human Bone Morphogenic Protein-2 | Medtronic | 7510200 | Clinically relevant treatment used as positive control |
| Isoflurane | Clipper | 10250 | Anesthesia for rats |
| IVIS | Perkin Elmer | 124262 | Bioluminescence imaging modality |
| Jig | Custom | Used to place bicortical pins | |
| Lipofectamine MessengerMAX | Fisher Scientific | LMRNA003 | mRNA complexing agent that enables mRNA delivery |
| Sensorcaine-MPF (Bupivicane (0.25%) and Epinephrine (1:200,000)) | APP Pharmaceuticals, LLC | NDC 63323-468-37 | Applied to surgical site for pain relief and vasoconstriction |
| Sterile water | Hospira | 8904653 | Used as solvent for cefazolin powder |
| Titanium external fixator plates | Custom | Prepared in house with scrap titanium and milling machine | |
| Total Performance System (TPS) Console | Stryker | OR-S-5100-1 | Used to create 5mm defect in femur |
| TPS MicroSaggital Saw | Stryker | OR-S-5100-34 | Used to create 5mm defect in femur |
| Ultrafocus Faxitron with DXA | Faxitron | High resolution radiographic imaging modality | |
| Uniprim rat diet | Envigo | TD.06596 | Medicated rat diet |
| Universal Handswitch for TPS | Stryker | OR-S-5100-9 | Used to create 5mm defect in femur |
| Vetbond Tissue Adhesive | 3M | 1469 | Skin closure |
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Citar este artigo
Kerzner, B., Martin, H. L., Weiser, M., Fontana, G., Russell, N., Murphy, W. L., Lund, E. A., Doro, C. J. A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator. J. Vis. Exp. (145), e59206, doi:10.3791/59206 (2019).