开发Wistar白化大鼠股骨截骨模型的协议
1Department of Orthopedics,All India Institute of Medical Sciences (AIIMS), 2Department of Biochemistry,All India Institute of Medical Sciences (AIIMS), 3Department of Anaesthesiology Pain Medicine and Critical Care,All India Institute of Medical Sciences (AIIMS), 4Department of Radiodiagnosis,All India Institute of Medical Sciences (AIIMS), 5Department of Pathology,All India Institute of Medical Sciences (AIIMS), 6Central Council for Research in Homoeopathy (CCRH)
Summary
在这里，我们提出了一种医源性骨折Wistar白化大鼠股骨干并跟进愈伤组织发展的协议。这种股骨截骨模型可以帮助研究人员评估骨折愈合的过程，并研究药物如何影响骨折愈合。
Abstract
骨折愈合是通过成骨细胞和破骨细胞的协调作用导致骨缺损再生的生理过程。骨合成代谢药物有可能增强骨折的修复，但具有高成本或不良副作用等限制。药物的骨愈合潜力最初可以通过 体外 研究来确定 ，但最终 的概念验证需要体内研究。我们的目标是开发一种股骨截骨啮齿动物模型，该模型可以帮助研究人员了解股骨干骨折后愈伤组织形成的发展，并有助于确定潜在的药物是否具有骨愈合特性。成年雄性Wistar白化大鼠在机构动物伦理委员会批准后使用。对啮齿动物进行麻醉，在无菌条件下，使用开放截骨术在股骨干中部三分之一处产生完全横向骨折。骨折复位并使用髓内K线进行内部固定，并允许进行继发性骨折愈合。术后给予腹膜内镇痛药和抗生素5 d。每周连续 X 线检查评估愈伤组织形成情况。根据放射学预先确定的时间点处死大鼠，放射学和免疫组织化学分析骨折愈伤组织的发展。
Introduction
骨是一种致密的结缔组织，由成骨细胞、成骨细胞和骨吸收细胞（破骨细胞）组成。骨折愈合是通过成骨细胞和破骨细胞的协调作用导致骨缺损再生的生理过程1.当发生骨折时，骨折部位的成骨细胞和破骨细胞活动是决定骨愈合的一些重要因素2。当骨折愈合偏离其正常病程时，会导致愈合延迟、畸形愈合或不愈合。当骨折愈合失败 9 个月，最近 3 个月内没有修复进展时，称为骨折不愈合3.大约 10%-15% 的骨折修复延迟，可能进展为不愈合4。所有骨折的不愈合率为 5%-10%，并且因受累骨骼和骨折部位而异5.
目前治疗骨折不愈合的方案包括手术和/或药物方式。目前，骨折延迟或不愈合可以通过骨移植等手术策略来克服。然而，骨移植有其局限性和并发症，如移植组织的可用性、供体部位疼痛、发病率和感染6。药物治疗包括骨合成代谢药物，如骨形态发生蛋白（BMP）和特立帕肽（副激素类似物）。目前使用的骨合成代谢药物有可能增强骨折的修复，但具有诸如高昂的成本或不良副作用等限制7。因此，有确定具有成本效益的非手术骨愈合替代方案的空间。药物的骨愈合潜力最初可以通过 体外 研究来确定 ，但最终 的概念验证需要体内研究。已知可增强骨愈合的药物应在 体外 进行评估，如果发现有希望，可用于 体内 动物模型研究。如果药物在 体内 模型中被证明可以促进骨形成和重塑，则可以进入下一阶段（即临床试验）。
评估动物的骨折愈合是评估在进行人体试验之前引入骨愈合的新型药物的合乎逻辑的一步。对于骨折愈合的 体内 动物模型研究，啮齿动物已成为越来越流行的模型8。由于运营成本低，对空间的需求有限，骨骼愈合所需的时间更少，啮齿动物模型引起了越来越多的兴趣9。此外，啮齿动物具有广泛的抗体和基因靶标，可以研究骨愈合和再生的分子机制10。共识会议全面强调了各种小动物骨骼愈合模型，重点关注影响骨骼愈合的不同参数，并强调了几种小动物骨折模型和植入物11。
基本裂缝模型大致可分为开放模型或封闭模型。闭合性骨折模型在骨骼上使用三点或四点弯曲力，不需要传统的手术方法。它们导致斜骨折或螺旋骨折，类似于人类的长骨折，但骨折位置和尺寸缺乏标准化可能是其中的混杂因素12。开放性骨折模型需要手术通路才能进行骨切开术，有助于在骨折部位实现更一致的骨折模式，但与闭合模型相比，与愈合延迟有关13。用于研究骨折愈合的骨骼选择主要仍然是胫骨和股骨，因为它们的尺寸和可及性。骨折部位的选择通常是骨干或干骺端。在骨质疏松症受试者研究中研究骨折愈合的情况下，专门选择干骺端区域，因为干骺端受骨质疏松症的影响更大14.可以使用髓内销和外固定器等几种植入物来稳定骨折11，15。
这项研究的目的是开发一种简单易懂的啮齿动物模型，该模型不仅可以帮助研究人员了解股骨骨折后愈伤组织的发展，还可以通过了解其作用机制来帮助确定潜在药物是否具有骨愈合特性。
Protocol
动物实验是在获得印度新德里AIIMS机构动物伦理委员会（IAEC）的伦理批准后进行的（286/IAEC-1/2021）。 1.术前程序 家养雄性Wistar白化大鼠6-8周龄，每只体重在150-200克之间，在中央动物设施（CAF）的单独笼子里。这确保了当多只大鼠共用笼子时不会造成手术/骨折部位损伤。 将大鼠保持在相对湿度为50%±5%的湿度控制环境中的温度为23°C±2°C，将它们暴?…
Representative Results
本研究旨在开发Wistar白化大鼠的股骨截骨模型。该模型可用于评估骨愈合，以及有前途的骨合成代谢药物在骨愈合中的成骨作用。遵循标准的手术预防措施和方案。手术使用了无菌长袍，窗帘和手术设备（图1）。设备（表1）在手术前48小时灭菌。按照协议使用麻醉剂，镇痛剂和抗生素，以确保动物始终保持无疼痛和感染。可以从每只大鼠的尾静脉收集血液（0.5m…
Discussion
该方法清晰地描述了在Wistar白化大鼠中开发骨折截骨模型所需的细节。该模型可用于评估有前途的骨合成代谢药物在骨折愈合中的成骨作用，以及了解骨愈合的复杂性。这种方法的显着特点是简单，不需要太多时间或复杂的设备。该方法选取成年雄性白化大鼠作为实验的啮齿动物模型。选择统一的性别以消除与性激素相关的骨骼愈合的任何混杂因素。
这项研究遵循开放截骨?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢印度政府AYUSH部顺势疗法研究中央委员会（CCRH）的研究经费。作者感谢新德里AIIMS中央动物设施的帮助和支持，感谢他们对动物实验的帮助和支持，感谢新德里AIIMS的CMET，感谢他们在摄影和摄像方面的帮助和支持。
Materials
| Alcohol | Raman & Weil Pvt. Ltd, Mumbai, Maharashtra, India | MFG/MD/2019/000189 | Sterillium hand disinfectant |
| Artery forceps | Nebula surgical, Gujarat, India | G.105.05S | 5", straight |
| Bard-Parker handle | Nebula surgical, Gujarat, India | G.103.03 | Size number 3 |
| Betadine solution | Win-medicare New Delhi, India | UP14250000001 | 10% w/v Povidone iodine solution |
| Cat's-paw skin retractor | Nebula surgical, Gujarat, India | 908.S | Small |
| EDTA | Sisco research laboratories Pvt. Ltd, Maharashtra, India | 43272 | Disodium salt |
| Eosin | Sigma Aldrich, Merck Life Sciences Pvt Ltd, Mumbai, Maharashtra, India | 115935 | For preparing the staining solution |
| Forceps (plain) | Nebula surgical, Gujarat, India | 115.06 | 6", plain |
| Forceps (toothed) | Nebula surgical, Gujarat, India | 117.06 | 6", toothed |
| Formaldehyde | Sisco research laboratories Pvt. Ltd, Maharashtra, India | 84439 | For preparing the neutral buffered formalin |
| Haematoxylin | Sigma Aldrich, Merck Life Sciences Pvt Ltd, Mumbai, Maharashtra, India | 104302 | For preparing the staining solution |
| Hammer | Nebula surgical, Gujarat, India | 401.M | |
| Injection Cefuroxime | Akumentis Healthcare Ltd, Thane, Maharashtra, India | 48/UA/SC/P-2013 | Cefuroxime sodium IP, 1.5 g/vial |
| Injection Ketamine | Baxter Pharmaceuticals India Private Limited, Gujarat, India | G/28-B/6 | Ketamine hydrochloride IP, 50 mg/mL |
| Injection Xylazine | Indian Immunologicals Limited, Hyderabad, Telangana, India | 28/RR/AP/2009/F/G | Xylazine hydrochloride USP, 20 mg/mL |
| Injection Lignocaine | Jackson laboratories Pvt Limited, Punjab, India | 1308-B | 2% Lignocaine Hydrochloride IP, 21.3 mg/mL |
| Injection Tramadol | Intas Pharmaceuticals Limited, Ahmedabad, Gujarat, India | MB/07/500 | Tramadol hydrochloride IP, 50 mg/mL |
| K-wire | Nebula surgical, Gujarat, India | 166 (1mm) | 12", double ended |
| Mechanical drill for inserting K-wire | Bosch, Germany | 06019F70K4 | GSR 120-LI Professional |
| Metzenbaum cutting scissors | Nebula surgical, Gujarat, India | G.121.06S | 6", straight |
| Needle holder | Nebula surgical, Gujarat, India | G.108.06 | 6", straight |
| Ophthalmic ointment | GlaxoSmithKline Pharmaceutical Limited, Bengaluru, Karnataka, India | KTK/28a/467/2001 | Neomycin, Polymixin B sulfate and Bacitracin zinc ophthalmic ointment USP |
| Osteotome (chisel) | Nebula surgical, Gujarat, India | 1001.S.10 | 10 mm, straight |
| Periosteal elevator | Nebula surgical, Gujarat, India | 918.10.S | 10 mm, straight |
| Pliers cum wire cutter | Nebula surgical, Gujarat, India | 604.65 | |
| Reynold’s scissors | Nebula surgical, Gujarat, India | G.110.06S | 6", straight |
| Standard semi-synthetic diet | Ashirvad Industries, Chandigarh, India | No catalog number available | Detailed composition provided in materials used |
| Steel cup for keeping betadine for application | Local purchase | No catalog number available | |
| Steel tray with lid for autoclaving instruments | Local purchase | No catalog number available | |
| Sterile gauze | Ideal Healthcare Industries, Delhi, India | E(0047)/14/MNB/7951 | Sterile, 5cmx5cm, 12 ply |
| Sterile marble block for support | Local purchase | No catalog number available | Locally fabricated; autoclavable |
| Syringe and needle (1 mL) | Becton Dickinson India Pvt. Ltd., Haryana, India | REF 303060 | 1 mL sterile Syringe with 26 G x 1/2 (0.45 mm x 13 mm) needle |
| Syringe and needle (2 mL) | Becton Dickinson India Pvt. Ltd., Haryana, India | REF 307749 | 2 mL sterile syringe with 24 G x 1'' (0.55 mm x 25 mm) needle |
| Syringe and needle (10 mL) | Hindustan Syringes & Medical Devices Ltd. Faridabad, India | 334-B(H) | 10 mL sterile syringe with 21 G x1.5" (0.80 mm x 38 mm) needle |
| Surgical blades (size no.15) | Paramount Surgimed Ltd, New Delhi, India for Medline Industries Inc, IL, USA | REF MDS15115E | Sterile, Single use |
| Surgical blades (size no.24) | Paramount Surgimed Ltd, New Delhi, India for Medline Industries Inc, IL, USA | REF MDS15124E | Sterile, Single use |
| Sutures | Healthium Medtech Pvt Ltd, Bangalore, Karnataka, India | SN 3318 | 4-0, 16 mm, 3/8 circle cutting needle, monofilament polyamide suture |
| Wax block in aluminium tray | Locally fabricated | No catalog number available | 30 cm x 30 cm x 4 cm aluminium tray containing wax (to prevent animal from slipping) |
| X-ray machine | Philips India Ltd, Gurugram, Haryana | SN19861013 | Model: Philips Digital Diagnost R 4.2 |
References
- Wang, T., Zhang, X., Bikle, D. D. Osteogenic differentiation of periosteal cells during fracture healing. Journal of Cellular Physiology. 232 (5), 913-921 (2017).
- Fakhry, M., Hamade, E., Badran, B., Buchet, R., Magne, D. Molecular mechanisms of mesenchymal stem cell differentiation towards osteoblasts. World Journal of Stem Cells. 5 (4), 136-148 (2013).
- Bishop, J. A., Palanca, A. A., Bellino, M. J., Lowenberg, D. W. Assessment of compromised fracture healing. JAAOS – Journal of the American Academy of Orthopaedic Surgeons. 20 (5), 273-282 (2012).
- Fong, K., et al. Predictors of nonunion and reoperation in patients with fractures of the tibia: an observational study. BMC Musculoskeletal Disorders. 14 (1), 103 (2013).
- Ramoutar, D. N., Rodrigues, J., Quah, C., Boulton, C., Moran, C. G. Judet decortication and compression plate fixation of long bone nonunion: Is bone graft necessary. Injury. 42 (12), 1430-1434 (2011).
- Goulet, J. A., Senunas, L. E., DeSilva, G. L., Greenfield, M. L. V. H. Autogenous iliac crest bone graft: Complications and functional assessment. Clinical Orthopaedics and Related Research. 339, 76-81 (1997).
- Stevenson, M., et al. A systematic review and economic evaluation of alendronate, etidronate, risedronate, raloxifene and teriparatide for the prevention and treatment of postmenopausal osteoporosis. Health Technology Assessment. 9 (22), 1 (2005).
- Haffner-Luntzer, M., Kovtun, A., Rapp, A. E., Ignatius, A. Mouse models in bone fracture healing research. Current Molecular Biology Reports. 2 (2), 101-111 (2016).
- Mills, L. A., Simpson, A. H. R. W. In vivo models of bone repair. The Journal of Bone and Joint Surgery. British Volume. 94 (7), 865-874 (2012).
- Houdebine, L. -. M., Sioud, M. Transgenic Animal Models in Biomedical Research. Target Discovery and Validation Reviews and Protocols: Volume 1, Emerging Strategies for Targets and Biomarker Discovery. , (2007).
- Histing, T., et al. Small animal bone healing models: Standards, tips and pitfalls results of a consensus meeting. Bone. 49 (4), 591-599 (2011).
- Bonnarens, F., Einhorn, T. A. Production of a standard closed fracture in laboratory animal bone. Journal of Orthopaedic Research. 2 (1), 97-101 (1984).
- Klein, M., et al. Comparison of healing process in open osteotomy model and open fracture model: delayed healing of osteotomies after intramedullary screw fixation. Journal of Orthopaedic Research. 33 (7), 971-978 (2015).
- Kolios, L., et al. Do estrogen and alendronate improve metaphyseal fracture healing when applied as osteoporosis prophylaxis. Calcified Tissue International. 86 (1), 23-32 (2010).
- Holstein, J. H., et al. Advances in the establishment of defined mouse models for the study of fracture healing and bone regeneration. Journal of Orthopaedic Trauma. 23, 31-38 (2009).
- Umiatin, U., Dilogo, I. H., Sari, P., Wijaya, S. K. Histological analysis of bone callus in delayed union model fracture healing stimulated with pulsed electromagnetic fields (PEMF). Scientifica. 2021, 4791172 (2021).
- Han, W., et al. The osteogenic potential of human bone callus. Scientific Reports. 6, 36330 (2016).
- Haffner-Luntzer, M., et al. A novel mouse model to study fracture healing of the proximal femur. Journal of Orthopaedic Research. 38 (10), 2131-2138 (2020).
- Aurégan, J. C., et al. The rat model of femur fracture for bone and mineral research: An improved description of expected comminution, quantity of soft callus and incidence of complications. Bone & Joint Research. 2 (8), 149-154 (2013).
- Li, Z., Helms, J. A. Drill hole models to investigate bone repair. Methods in Molecular Biology. 2221, 193-204 (2021).
- Handool, K. O., et al. Optimization of a closed rat tibial fracture model. Journal of Experimental Orthopaedics. 5 (1), 13 (2018).
- Kobata, S. I., et al. Prevention of bone infection after open fracture using a chitosan with ciprofloxacin implant in animal model. Acta Cirurgica Brasileira. 35 (8), 202000803 (2020).
Cite This Article
Aryal, A., Pagaku, P. K., Dey, D., Tyagi, S., Shrivastava, V., Bhattacharya, A., Rani, S., Nayak, D., Khurana, A., Khanna, P., Goyal, A., Mridha, A. R., Garg, B., Sen, S. Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats. J. Vis. Exp. (186), e63712, doi:10.3791/63712 (2022).