兔子骨增增的千叶模型,用于骨代材料骨生长和新血管化的评估
1Division of Fixed Prosthodontics and Biomaterials, Biomaterials Laboratory,University of Geneva, University Clinic of Dental Medicine, 2Department of Surgery, Division of Oral and Maxillofacial Surgery (HUG), Unity of Oral Surgery and Implantology,University of Geneva, University Clinic of Dental Medicine, 3Department of Surgery, Division of Oral and Maxillofacial surgery (HUG), Laboratory of Oral & Maxillofacial Pathology,University of Geneva, University Clinic of Dental Medicine, 4Division of Fixed Prosthodontics and Biomaterials,University of Geneva, University Clinic of Dental Medicine
Summary
在这里,我们提出了一个手术方案在兔子,目的是评估骨替代材料的骨再生能力。通过使用固定在兔子头骨上的PEEK圆柱体,可以对活体动物或安乐死动物进行骨传导、骨诱导、成骨和血管生成。
Abstract
兔钙质模型的基本原理是在颅骨皮质部分垂直生长新的骨组织。该模型允许评估骨替代材料的口腔和颅骨再生在骨生长和新血管化支持方面。一旦动物被麻醉和通风(内切管插管),四个圆柱体由圆柱体醚酮(PEEK)拧到头骨上,在中位和日冕缝合线两侧。在由每个圆柱体划定的骨骼区域内钻出五个髓洞,允许骨髓细胞的流入。材料样品被放入气缸中,然后关闭。最后,手术部位被缝合,动物被唤醒。骨骼生长可以通过微断层扫描在活体动物身上评估。一旦动物被安乐死,骨骼生长和新血管化可以通过微断层扫描、免疫组织学和免疫荧光进行评估。由于材料评估需要最大的标准化和校准,因此千数模型显得非常理想。检修非常简单,使用定义的气缸可促进校准和标准化,并可同时评估四个样品。此外,可以使用活断层扫描,最终有望大量减少要安乐死的动物。
Introduction
骨增增的千数模型是在90年代发展起来的,目的是优化口腔和颅面外科领域的引导性骨再生(GBR)概念。该模型的基本原理是在颅骨皮质部分垂直生长新的骨组织。为此,将反应器(如钛圆顶、圆柱或-笼)固定在头骨上,以保护移植物(如水凝胶、骨代用品等)进行的骨再生。在这个模型的帮助下,钛或陶瓷保持架1,2,3,4,5,6,GBR膜7,8,9 ,10, 骨原因子11,12,13,14,15,16,17,新骨替补12,16,17,18,19,20,21,22,23,24,25,26,27,28,29或骨再生过程中新血管化机理30被评估。
从平移的角度来看,千数模型表示一个单壁缺陷,可以与钳口31中的IV类缺陷进行比较。目标是在皮质区域上方生长新骨骼,无需内源性骨壁的任何横向支撑。因此,该模型非常严格,并评估了垂直骨传导在骨皮部分的实际潜力。如果本文描述的模型主要用于评估骨代用品中的骨传导,则还可以评估成骨和/或骨诱导,以及血管生成1、2、3、 4,5,6,7,8,9,10,11,12,13 ,14,15,16,17,18,19,20,21,22 ,23,24,25,26,27,28,29,30 。
基本上出于伦理、实际和经济原因,在兔子中发展了钙化模型,其中骨代谢和结构与人类32相比相当相关。在上述30个参考文献中,80%使用兔钙模式1,2,3,4,5,6,7,8 ,9,10,11,12,13,14,15,17,22, 23,26,27,28,29,30,33,从而证明了这种动物模型的相关性。2008年,Busenlechner小组将骨质模型转移到了猪,允许同时比较8种骨代用品20个(与兔子的两个骨代用品相比)。另一方面,我们小组将兔肉模型转移到绵羊身上。简而言之,钛圆顶被放置在羊头骨上,以描述一种新的3D打印骨替代品的骨传导。这些研究使我们能够开发和掌握千数模型及其分析16,21。
最后三项研究引用了16、20、21,以及其他几项调查12、17、18、19、22 23,24,26,27,28,29,证实了千花模型的巨大潜力作为筛选和表征模型。然而,尽管获得的结果相当令人满意,他们也指出了一些局限性:(1)使用钛圆顶,防止了X射线扩散,反过来又活微CT使用。在组织学处理之前,这些样品无法去除,迫使研究人员将样品嵌入聚(甲基丙烯酸酯)树脂(PMMA)中。因此,由此产生的分析主要限于地形。(2) 高昂的财务费用,特别是因为动物的费用,以及与动物的后勤、维护和手术有关的费用。(3) 难以获得大型动物的道德批准。
Polo等人最近进行的一项研究在很大程度上改进了兔子的模型。钛圆顶被可堵塞的圆柱体所取代,这些圆筒可以填充恒定的材料量。其中四个圆柱体被放置在兔子头骨上。完成后,可以去除钢瓶,使活检无金属,在样品处理方面引入更大的灵活性。兔钙模型在成本更低、动物处理方便、样品处理方便的同时,对同时测试极具吸引力。利用这些最近的发展,我们进一步改善了模型,用PEEK取代钛来生产圆柱体,从而允许X射线扩散和在活体动物身上使用微断层扫描。
在本文中,我们将描述麻醉和手术过程,并展示使用该协议可以获得的输出示例,即(免疫-)组织学、组织学、活体和外体微断层扫描,以评估骨骼机制再生和量化由骨替代材料支持的新骨合成。
Protocol
根据瑞士的法律要求,该协议由一个学术委员会批准,并由州和联邦兽医机构监督(批准程序为 GE/165/16 和 GE/100/18)。 1. 特定设备和动物 缸 PEEK 外侧稳定卡舌的机器油缸内径为 5 mm,外径为 8 mm,高度为 5 mm(图 1)。 机器 PEEK 盖的设计允许精确地夹在气缸顶部(厚度为 1 mm)。 手术前通过高压灭菌对PEEK气缸和瓶盖进行消?…
Representative Results
本文描述的模型专门用于骨代用品中骨传导的评估。骨代生和骨代用品的骨质诱导(预)细胞或加载生物活性分子也可以评估,以及血管发生1,2,3,45,6,7,8,…
Discussion
本文描述的模型很简单,只要遵循所有步骤,设备合适,就很容易开发。由于所述方案是一种手术方法,所有步骤都显得至关重要,必须正确遵循。对动物实验进行训练至关重要,特别是在兔子处理和麻醉方面。不要犹豫,要求专业的麻醉和兽医帮助。坚持在缝合切除前后对动物进行日常视觉监测至关重要。即使头骨的皮肤厚、丰、松,气瓶的固定也会引起较大的张力。如果缝合线过早切除,伤口可能会重新?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢Geistlich AG(沃尔胡森,CH)和骨病学基金会(卢塞恩,CH)(授予n_18-049)的支持,以及全球D(布里格奈斯,FR)提供螺丝。特别感谢盖斯特利希的B.舍费尔博士。我们也感谢埃利亚内·杜布瓦和克莱尔·赫尔曼的出色组织学处理和他们的宝贵建议。最后,我们热烈感谢哈维尔·贝林、西尔维·轮盘和Pr Walid Habre的整个团队,”实验性手术Dpt”,感谢他们出色的技术援助。
Materials
| Drugs | |||
| Enrofloxacine Baytril 10% | Bayer | Antibiotic | |
| Fentanyl | Bischel | For analgesia | |
| Ketalar 50mg/ml | Pfizer | Ketamine for anesthesia | |
| Lidohex | Bichsel | Lubricating gel for the eyes | |
| Opsite | Smith and Nephew | 66004978 | Sprayable dressing |
| Povidone iodine 10%, Betadine | Mundipharma | anti-infective agent | |
| Propofol 2% | Braun | 3538710 | For anesthesia |
| Rapidocain 2% | sintetica | Local anesthesia | |
| Ringer-acetate | Fresenius Kabi | Volume compensation | |
| Rompun 2% | Bayer | Xylazin for anesthesia | |
| Sevoflurane 5% | Abbvie | For anesthesia | |
| Sterile saline | Sintetica | ||
| Temgesic | Reckitt Benckiser | Buprenorphine hydrochloride, analgesia | |
| Thiopental Inresa | Ospediala | For anesthesia | |
| Xylocaine 10% spray | Astra Zeneca | For intubation | |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Fresenius Vial pilot C | Imexmed | Infusion pump | |
| Heated pad | Harvard Apparatus | ||
| Suction dominant 50 | Medela | ||
| Suction tubing Optimus | Promedical | 80342.2 | |
| Surgical motor | Schick dental | Qube | Drilling of intramedullary holes |
| Ventilation | Maquet Servo1 | ||
| Name | Company | Catalog Number | Comments |
| Material | |||
| Cylinders and caps | Boutyplast | Customized | composition: PEEK (poly ether ether ketone) |
| Manual self-retaining shaft | GlobalD | ACT1K | |
| Mobile handle for self-retaining shaft | GlobalD | MTM | |
| Self- drilling screws | GlobalD | VA1.2KL4 | cross-drive screws composed by Titanium grade5, ISO 5832-3 |
| Name | Company | Catalog Number | Comments |
| Surgical tray | |||
| Endotracheal tube Shiley diameter 2,5mm | Covidien | 86233 | For intubation |
| Endotracheal tube Shiley diameter 4,9mm | Covidien | 107-35G | For intubation |
| Ethicon prolene 4-0 | Ehticon | 8581H | Non-resorbable suture |
| Forceps | Marcel Blanc | BD027R | 145 mm |
| Intubation catheter | Cook medical | Guide for intubation | |
| Needlle holder | Marcel Blanc | BM008R | |
| Needles BD Microlance3 | Becton Dickinson | 300300/304622 | 26G; 18G |
| Periosteal | HU-Friedy | P9X | |
| Round surgical burs | Patterson | 78000 | 0.8 mm in diameter, Drilling of intramedullary holes |
| Scalpel | Swann-Morton | n°10 and n°15 | |
| Scissors | Marcel Blanc | 00657 | 180 mm |
| Syringes Omnifix | Braun | 4616057V | 5ml, 10ml and 50ml |
| Venflon G22 | Braun | 42690985-01 | Vasofix safety for the ear iv line |
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Cite This Article
Marger, L., Barone, A., Martinelli-Kläy, C. P., Schaub, L., Strasding, M., Mekki, M., Sailer, I., Scherrer, S. S., Durual, S. Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials. J. Vis. Exp. (150), e59976, doi:10.3791/59976 (2019).