大鼠胫骨生长板损伤模型，用于表征修复机制并评估生长板再生策略
1Department of Bioengineering, Department of Orthopedics,University of Colorado Anschutz Medical Campus, 2Department of Orthopedics,University of Colorado Anschutz Medical Campus, 3Department of Chemical & Biological Engineering,Colorado School of Mines, 4Department of Orthopedics,Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus
Summary
生长板是发生纵向生长的儿童长骨中的软骨区域。当受伤时，骨组织可能形成并损害生长。我们描述了生长板损伤的大鼠模型，其导致骨修复组织，允许研究修复机制和生长板再生策略。
Abstract
所有儿科骨折的三分之一涉及生长板，并可能导致骨生长受损。生长板（或分裂）是在所有长骨结束时发现的软骨组织，其负责纵向骨生长的儿童。一旦损伤，生长板内的软骨组织就可能发生过早的骨化，并导致不必要的骨修复组织，形成“骨棒”。在一些情况下，这种骨棒可能导致骨生长畸形，例如角度畸形，或者可以完全停止纵向骨生长。目前没有临床治疗可以完全修复受伤的生长板。使用生长板损伤的动物模型更好地了解骨棒形成的机制，并确定抑制它的方法是开发更好的生长板损伤治疗的好机会。该协议描述了如何使用钻孔缺陷来破坏大鼠近端胫骨生长板。这个sma动物模型可靠地产生一个骨棒，并可能导致类似于在儿童中看到的生长畸形。该模型允许调查骨条形成的分子机制，并且作为测试生长板损伤的潜在治疗选择的手段。
Introduction
生长板损伤占所有儿科骨折的30％，可能导致骨生长受损1 。除骨折外，生长板受伤可能由其他病因引起，包括骨髓炎2 ，原发性骨肿瘤3 ，放射和化学疗法4以及医源性损伤5 。生长板（或Physis）是儿童长骨结束时的软骨区域，负责纵向骨生长。它通过软骨内骨化促进骨伸长;软骨细胞经历增殖和肥大，然后由进入的成骨细胞重塑以形成小梁骨6 。生长板也是发育骨架的弱区，容易受伤。生长板骨折或损伤的主要问题是生长板内损伤的软骨组织可以be替换为不需要的骨修复组织，也被称为“骨棒”。根据其生长盘中的大小和位置，骨棒可能导致角质畸形或完全生长停滞，对于尚未达到其全身高度的幼儿来说，这是一个破坏性的后遗症7 。
目前没有治疗可以完全修复受伤的生长板。一旦骨髓形成，临床医生必须决定是否手术切除它8 。至少2年或2厘米骨骼生长剩余的患者和跨越小于生长板面积的50％的骨棒通常是骨髓切除术的候选者8 。骨髓移植通常是随机插入自体脂肪移植物，以防止骨组织的重组，并允许周围的未受伤的生长板恢复生长。然而，这些技术是问题激素经常失败，导致骨条复发，对生长持续负面影响9 。迫切需要开发有效的治疗方法，不仅可以防止骨形成，还可以使生长板软骨再生，从而恢复正常的骨伸长。
骨棒形成的分子机制尚未完全阐明。对这些生物学机制的更多了解可以为受损生长板损伤的儿童带来更有效的治疗干预措施。由于在人类中研究这些机制是困难的，所以已经使用动物模型，特别是生长板损伤的大鼠模型10,11,12,13,14,15,16 。本文介绍的方法论文描述了大鼠胫骨生长板中的钻孔缺陷如何导致可预测和可重复的修复组织，其早于损伤后7天开始骨化，并且在损伤后28天形成完全成熟的骨块，重塑。这提供了一种小型动物体内模型，其中研究骨形成的生物学机制，以及评估可以预防骨棒和/或再生生长板软骨的新疗法。例如，该模型可用于测试可以再生生长板软骨的软骨形成生物材料，并为患有生长板损伤的儿童提供有价值的治疗。本文介绍的技术将描述用于产生生长板损伤的手术方法和随后将生物材料递送至损伤部位。我们还将讨论评估骨棒形成和修复组织的方法。
Protocol
所有动物手术必须得到当地机构动物保护和使用委员会（IACUC）的批准。以下程序的动物方案由科罗拉多大学丹佛IACUC批准。 获得大鼠注意：除非需要转基因动物，否则在手术时需要6周龄的骨骼不成熟的Sprague-Dawley大鼠。可能使用其他菌株;然而，大多数已发表的研究已经在Sprague-Dawley大鼠上进行。 2.手术用品的准备高压灭菌手?…
Representative Results
使用该方法成功的生长板损伤涉及胫骨生长板的中心的破坏而不破坏关节软骨表面。据报道，骨修复组织在损伤后约7天开始，并在损伤后28天完全发育，通过微型计算机断层扫描（micro CT）显现（ 图2 ）。虽然这些时间点在这里被选择以显示基于先前公布的数据的骨形成的开始和成熟，但是可以使用其他时间点来研究修复过程的各个阶段，从术后第1至6个月<sup…
Discussion
增长板损伤动物模型大大增加了我们对这种损伤的生物学机制的理解，从而潜在地导致对患有生长板损伤的儿童的更有效的治疗干预。为了成功创建一个骨棒，并使用本工作中提出的模型研究其体内形成，关键是通过钻孔到足够的深度而不破坏关节软骨来破坏生长板。动物的手术实施变化，以及在较小程度上解剖标志物的变化可能会导致问题的结果。我们建议执行上述关于尸体动物?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者承认美国国家卫生研究院（NIH）国家关节炎和肌肉骨骼和皮肤病国家研究所的资助，授权号为R03AR068087，科罗拉多大学医学院学术浓缩基金和盖茨再生医学中心。这项工作也得到NIH / NCATS科罗拉多CTSA授权号UL1 TR001082的支持。内容是作者的唯一责任，不一定代表NIH的正式观点。
Materials
| Scalpel handle | McKesson | MCK42332500 | |
| Needle holder | Stoelting | RS-7824 | |
| Adson tissue forceps | Sklar | 50-3048 | |
| Iris Scissors | Sklar | 47-1246 | |
| Rotary Tool | Dremel | 7700 | Variable speed rotary tool |
| Keyless Rotary Tool Chuck | Dremel | 4486 | |
| Dental Burs | Dental Burs USA | FG6 | Round carbide bur, ≤2mm |
| Steinmann pins | Simpex Medical | T-078 | |
| Hair clippers | Wahl | 5537N | |
| 3-0 PGA surutes | Oasis | MV-J398-V | |
| Sterile gauze 2×2" | Covidien | 441211 | |
| Povidone Iodine | McKesson | 922-00801 | |
| Sterile saline | Vetone | 510224 | |
| 10 ml luer lock syringe | Becton Dickinson | 309604 | |
| 23 gauge needle | Becton Dickinson | 305145 | |
| Isopropyl alcohol pads | Dynarex | 1113 | |
| Isoflurane | IsoFlo | 30125-2 | |
| Caliper | Mitutoyo | 500-196-30 | |
| Carprofen | Rimadyl | 27180 | |
| Buprenorphine | Par Pharmaceuticals Inc | NDC 42023-179 | |
| Fenestrated Surgical Drape | McKesson | 25-517 | |
| Surgical Gloves | Uline | S-20204 | |
| #15 Scalpel Blade | Aven | 44044 | |
| 9mm wound clips | Fine Science Tools | 12032-09 | |
| Reflex clip applier | World Precision Instruments | 500345 | |
| Absorbant underpads | McKesson | MON 43723110 | |
| Tec 3 Iso Vaporizer | VetEquip | 911103 | |
| Germinator 500 | Braintree Scientific | GER 5287-120V | |
| Warm water recirculator | Kent Scientific | TP-700 | |
| Absorbent Underpads | Medline Industries | MSC281230 |
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Cite This Article
Erickson, C. B., Shaw, N., Hadley-Miller, N., Riederer, M. S., Krebs, M. D., Payne, K. A. A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies. J. Vis. Exp. (125), e55571, doi:10.3791/55571 (2017).