Name: a reliable and reproducible critical-sized segmental femoral defect model in rats stabilized with a custom external fixator
Uploaded: 2019-03-24T15:00:00
Description: Watch this Scientific Journal Video about A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator at JoVE.com

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。

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
<ol><li>使用超繁殖的 Sprague-Dawley 雄性大鼠, 重约350克。</li>
</ol>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?...

<ol>
	<li>Filipowska, J., Tomaszewski, K. A., Nied&#378;wiedzki, &. #. 3. 2. 1. ;., Walocha, J. A., Nied&#378;wiedzki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+vasculature+in+bone+development,+regeneration+and+proper+systemic+functioning.">The role of vasculature in bone development, regeneration and proper systemic functioning.</a> Angiogenesis. 20 (3), 291-302 (2017).</li><li>Charalambous, C. P., Akimau, P., Wilkes, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+monolateral-ring+fixator+for+bone+transport+in+post-traumatic+femoral+segmental+defect:+A+technical+note.">Hybrid monolateral-ring fixator for bone transport in post-traumatic femoral segmental defect: A technical note.</a> Archives of Orthopaedic and Trauma Surgery. 129 (2), 225-226 (2009).</li><li>Xing, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+bilateral+femoral+large+segmental+bone+defect+mouse+model+potentially+applicable+to+basic+research+in+bone+tissue+engineering.">Establishment of a bilateral femoral large segmental bone defect mouse model potentially applicable to basic research in bone tissue engineering.</a> The Journal of Surgical Research. 192 (2), 454-463 (2014).</li><li>Chadayammuri, V., Hake, M., Mauffrey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+strategies+for+the+management+of+long+bone+infection:+A+review+of+the+Masquelet+technique.">Innovative strategies for the management of long bone infection: A review of the Masquelet technique.</a> Patient Safety in Surgery. 9 (32), (2015).</li><li>Koettstorfer, J., Hofbauer, M., Wozasek, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+limb+salvage+using+the+two-staged+technique+with+internal+fixation+after+osteodistraction+in+an+effort+to+treat+large+segmental+bone+defects+in+the+lower+extremity.">Successful limb salvage using the two-staged technique with internal fixation after osteodistraction in an effort to treat large segmental bone defects in the lower extremity.</a> Archives of Orthopaedic and Trauma Surgery. 132 (19), 1399-1405 (2012).</li><li>Fragomen, A. T., Rozbruch, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanics+of+external+fixation.">The mechanics of external fixation.</a> The Musculoskeletal Journal of Hospital for Special Surgery. 3 (1), 13-29 (2007).</li><li>O&#8217;Toole, R. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective+randomized+trial+to+assess+fixation+strategies+for+severe+open+tibia+fractures:+Modern+ring+external+fixators+versus+internal+fixation+(FIXIT+Study).">A prospective randomized trial to assess fixation strategies for severe open tibia fractures: Modern ring external fixators versus internal fixation (FIXIT Study).</a> Journal of Orthopaedic Trauma. 31, S10-S17 (2017).</li><li>F&#252;rmetz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+transport+for+limb+reconstruction+following+severe+tibial+fractures.">Bone transport for limb reconstruction following severe tibial fractures.</a> Orthopedic Reviews. 8 (1), 6384 (2016).</li><li>Dohin, B., Kohler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Masquelet&#8217;s+procedure+and+bone+morphogenetic+protein+in+congenital+pseudarthrosis+of+the+tibia+in+children:+A+case+series+and+meta-analysis.">Masquelet&#8217;s procedure and bone morphogenetic protein in congenital pseudarthrosis of the tibia in children: A case series and meta-analysis.</a> Journal of Children's Orthopaedics. 6 (4), 297-306 (2012).</li><li>Einhorn, T. A., Gerstenfeld, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing:+Mechanisms+and+interventions.">Fracture healing: Mechanisms and interventions.</a> Nature Reviews Rheumatology. 11, 45-54 (2015).</li><li>Pascher, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+to+cartilage+defects+using+coagulated+bone+marrow+aspirate.">Gene delivery to cartilage defects using coagulated bone marrow aspirate.</a> Gene Therapy. 11 (2), 133-141 (2004).</li><li>Glatt, V., Matthys, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjustable+stiffness,+external+fixator+for+the+rat+femur+osteotomy+and+segmental+bone+defect+models.">Adjustable stiffness, external fixator for the rat femur osteotomy and segmental bone defect models.</a> Journal of Visualized Experiments. (92), (2014).</li><li>Betz, O. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+percutaneous+gene+delivery+to+enhance+healing+of+segmental+bone+defects.">Direct percutaneous gene delivery to enhance healing of segmental bone defects.</a> The Journal of Bone and Joint Surgery. 88 (2), 355-365 (2006).</li><li>Fang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+new+bone+formation+by+direct+transfer+of+osteogenic+plasmid+genes.">Stimulation of new bone formation by direct transfer of osteogenic plasmid genes.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (12), 5753-5758 (1996).</li><li>Kaspar, K., Schell, H., Toben, D., Matziolis, G., Bail, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+easily+reproducible+and+biomechanically+standardized+model+to+investigate+bone+healing+in+rats,+using+external+fixation.">An easily reproducible and biomechanically standardized model to investigate bone healing in rats, using external fixation.</a> Biomedizinische Technik. 52 (6), 383-390 (2007).</li><li>Leary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AVMA+guidelines+for+the+euthanasia+of+animals:+2013+edition.">AVMA guidelines for the euthanasia of animals: 2013 edition.</a> American Veterinary Medical Association. , (2013).</li><li>McKay, W. F., Peckham, S. M., Badura, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+clinical+review+of+recombinant+human+bone+morphogenetic+protein-2+(INFUSE+Bone+Graft).">A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE Bone Graft).</a> International Orthopaedics. 31 (6), 729-734 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Living lmage Software. , (2006).</li><li>Bassett, J. H. D., Van Der Spek, A., Gogakos, A., Williams, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+X-ray+imaging+of+rodent+bone+by+faxitron.">Quantitative X-ray imaging of rodent bone by faxitron.</a> Methods in Molecular Biology. , 499-506 (2012).</li><li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+bone+healing+models:+Standards,+tips,+and+pitfalls+results+of+a+consensus+meeting.">Small animal bone healing models: Standards, tips, and pitfalls results of a consensus meeting.</a> Bone. 49 (4), 591-599 (2011).</li><li>Lieberman, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+regional+gene+therapy+with+bone+morphogenetic+protein-2-producing+bone-marrow+cells+on+the+repair+of+segmental+femoral+defects+in+rats.">The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats.</a> The Journal of Bone and Joint Surgery. 81 (7), 905-917 (1999).</li><li>Tsuchida, H., Hashimoto, J., Crawford, E., Manske, P., Lou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+allogeneic+mesenchymal+stem+cells+repair+femoral+segmental+defect+in+rats.">Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats.</a> Journal of Orthopaedic Research. 21 (1), 44-53 (2003).</li><li>Jiang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+standardized+massive+bone+defect+model+in+rats+employing+an+internal+eight-hole+stainless+steel+plate+for+bone+tissue+engineering.">Novel standardized massive bone defect model in rats employing an internal eight-hole stainless steel plate for bone tissue engineering.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (4), 2162-2171 (2018).</li><li>Baltzer, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+enhancement+of+fracture+repair:+Healing+of+an+experimental+segmental+defect+by+adenoviral+transfer+of+the+BMP-2+gene.">Genetic enhancement of fracture repair: Healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene.</a> Gene Therapy. 7 (9), 734-739 (2000).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+defect+animal+models+for+testing+efficacy+of+bone+substitute+biomaterials.">Bone defect animal models for testing efficacy of bone substitute biomaterials.</a> Journal of Orthopaedic Translation. 3 (3), 95-104 (2015).</li></ol>

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...

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。

<table>
	<tbody>
		<tr>
			<td>0.9% Sterile Saline</td>
			<td>Baxter</td>
			<td>2F7124</td>
			<td>Used for irrigating wound and rehydration</td>
		</tr>
		<tr>
			<td>10% Iodine/Povidone</td>
			<td>Carefusion</td>
			<td>1215016</td>
			<td>Used to prep skin</td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>VWR</td>
			<td>89370094</td>
			<td>Used as fixative</td>
		</tr>
		<tr>
			<td>1mm non-threaded kirschner wire</td>
			<td>DePuy Synthes</td>
			<td>VW1003.15</td>
			<td>Sterilized, used for the most proximal pin</td>
		</tr>
		<tr>
			<td>1mm threaded kirschner wire</td>
			<td>DePuy Synthes</td>
			<td>VW1005.15</td>
			<td>Sterilized, used for the 3 most distal pin slots</td>
		</tr>
		<tr>
			<td>2x2 gauze</td>
			<td>Covidien</td>
			<td>4006130</td>
			<td>Sterilized, used to prep skin and absorb blood</td>
		</tr>
		<tr>
			<td>4-0 Vicryl Suture</td>
			<td>Ethicon</td>
			<td>4015304</td>
			<td>Used to close muscle and skin layers</td>
		</tr>
		<tr>
			<td>4-40 x 0.25&#34;,18-8 stainless steel button head cap screws</td>
			<td>Generic</td>
			<td></td>
			<td>External fixator assembly</td>
		</tr>
		<tr>
			<td>4200 Cordless Driver</td>
			<td>Stryker</td>
			<td>OR-S-4200</td>
			<td>Used to drill kirschner wires</td>
		</tr>
		<tr>
			<td>4x4 gauze</td>
			<td>Covidien</td>
			<td>1219158</td>
			<td>Sterilized, used to absorb blood</td>
		</tr>
		<tr>
			<td>70 % Ethanol</td>
			<td></td>
			<td></td>
			<td>Used to prep skin</td>
		</tr>
		<tr>
			<td>Baytril</td>
			<td>Bayer Healthcare LLC, Animal health division</td>
			<td>312.10010.3</td>
			<td>Added to water as an antibiotic</td>
		</tr>
		<tr>
			<td>Cefazolin</td>
			<td>Hikma Pharmaceuticals</td>
			<td>8917156</td>
			<td>Pre-op antibiotic</td>
		</tr>
		<tr>
			<td>CleanCap Gaussia Luciferase mRNA (5moU)</td>
			<td>TriLink Biotechnologies</td>
			<td>L-7205</td>
			<td>Modified mRNA encoding for Gaussia Luciferase, keep on ice during use</td>
		</tr>
		<tr>
			<td>Coelenterazine native</td>
			<td>NanoLight Technology</td>
			<td>303</td>
			<td>Substrate for Guassia Luciferase, used to assess luciferase activity in vivo</td>
		</tr>
		<tr>
			<td>Double antibiotic ointment</td>
			<td>Johnson &#38; Johnson consumer Inc</td>
			<td>8975432</td>
			<td>Applied to pin sites post-op as wound care</td>
		</tr>
		<tr>
			<td>Dual Cut Microblade</td>
			<td>Stryker</td>
			<td>5400-003-410</td>
			<td>Used to create 5mm defect in femur</td>
		</tr>
		<tr>
			<td>Ethylenediamine Tetraacetic Acid (EDTA)</td>
			<td>Fisher</td>
			<td>BP120-500</td>
			<td>Used to decalcify bone to prep for histology</td>
		</tr>
		<tr>
			<td>Extended Release Buprenorphine</td>
			<td>ZooPharm</td>
			<td></td>
			<td>Used as 3 day pain relief</td>
		</tr>
		<tr>
			<td>Fenestrated drapes</td>
			<td>3M</td>
			<td>1204025</td>
			<td>Used to establish sterile field</td>
		</tr>
		<tr>
			<td>Handpiece cord for TPS</td>
			<td>Stryker</td>
			<td>OR-S-5100-4N</td>
			<td>Used to create 5mm defect in femur</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>K&#38;H Pet Products</td>
			<td>121239</td>
			<td>Rat body temperature maintenance</td>
		</tr>
		<tr>
			<td>Hexagonal head screwdriver</td>
			<td>Wiha</td>
			<td>263/1/16 &#34; X 50</td>
			<td>External fixator tightening</td>
		</tr>
		<tr>
			<td>Induction chamber</td>
			<td>Generic</td>
			<td></td>
			<td>Anesthesia for rats</td>
		</tr>
		<tr>
			<td>Infuse collagen sponge with recombinant human Bone Morphogenic Protein-2</td>
			<td>Medtronic</td>
			<td>7510200</td>
			<td>Clinically relevant treatment used as positive control</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Clipper</td>
			<td>10250</td>
			<td>Anesthesia for rats</td>
		</tr>
		<tr>
			<td>IVIS</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>Bioluminescence imaging modality</td>
		</tr>
		<tr>
			<td>Jig</td>
			<td>Custom</td>
			<td></td>
			<td>Used to place bicortical pins</td>
		</tr>
		<tr>
			<td>Lipofectamine MessengerMAX</td>
			<td>Fisher Scientific</td>
			<td>LMRNA003</td>
			<td>mRNA complexing agent that enables mRNA delivery</td>
		</tr>
		<tr>
			<td>Sensorcaine-MPF (Bupivicane (0.25%) and Epinephrine (1:200,000))</td>
			<td>APP Pharmaceuticals, LLC</td>
			<td>NDC 63323-468-37</td>
			<td>Applied to surgical site for pain relief and vasoconstriction</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>Hospira</td>
			<td>8904653</td>
			<td>Used as solvent for cefazolin powder</td>
		</tr>
		<tr>
			<td>Titanium external fixator plates</td>
			<td>Custom</td>
			<td></td>
			<td>Prepared in house with scrap titanium and milling machine</td>
		</tr>
		<tr>
			<td>Total Performance System (TPS) Console</td>
			<td>Stryker</td>
			<td>OR-S-5100-1</td>
			<td>Used to create 5mm defect in femur</td>
		</tr>
		<tr>
			<td>TPS MicroSaggital Saw</td>
			<td>Stryker</td>
			<td>OR-S-5100-34</td>
			<td>Used to create 5mm defect in femur</td>
		</tr>
		<tr>
			<td>Ultrafocus Faxitron with DXA</td>
			<td>Faxitron</td>
			<td></td>
			<td>High resolution radiographic imaging modality</td>
		</tr>
		<tr>
			<td>Uniprim rat diet</td>
			<td>Envigo</td>
			<td>TD.06596</td>
			<td>Medicated rat diet</td>
		</tr>
		<tr>
			<td>Universal Handswitch for TPS</td>
			<td>Stryker</td>
			<td>OR-S-5100-9</td>
			<td>Used to create 5mm defect in femur</td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469</td>
			<td>Skin closure</td>
		</tr>
	</tbody>
</table>

a reliable and reproducible critical-sized segmental femoral defect model in rats stabilized with a custom external fixator

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。

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...

Watch this Scientific Journal Video about A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator at JoVE.com

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

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。

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

此手术方案使用一致且可重复的外部固定段缺陷模型来评估骨骼的愈合机制和潜在再生疗法的使用。我们精简了具有挑战性的手术，用于创建和稳定一个可访问且经济高效的部分骨缺陷模型，同时仍然适合纵向愈合研究。尽管该模型需要进一步研究，但该技术尤其适用于创伤性关键大小的糖尿病骨缺陷的治疗，这些缺陷可能受益于外部固定的稳定。为防止钻取针脚时出现断裂，请使用高旋转/分钟和低压刺穿侧皮层，然后减少每分钟的旋转以前进。在用骨质原体蛋白管骨移植试剂盒制备脚手架后，根据制造商的说明，使用无菌剪刀和无菌尺子修剪胶原蛋白海绵，以适合五由三毫米的缺陷。然后使用注射器将骨形态遗传蛋白-2溶液均匀地分布到海绵表面，直到整个溶液被吸收。接下来，用第13根肋骨、后爪和腹中线作为边缘，剃掉麻醉大鼠后腿周围的区域，然后用10%的波维酮碘和70%乙醇浸泡的无菌纱布连续擦洗暴露的皮肤。为了诱发股骨缺陷，将剃光的腿伸进一个细小的透明粘性窗帘，用无菌毛巾盖住手术台，形成无菌领域。剥皮股骨，并使用编号 15 刀片，通过从小股板延伸到近端股骨的较大肌酸酯的皮肤创建一个反边切口。小心地沿肌肉间隔膜将侧腿筋膜分开，以将前四头肌的广大后肌肌肉与腿筋后部分离，直到侧股骨暴露。保持15号手术刀刀片平行于骨骼表面的轮廓，轻轻地将肌肉从底层骨骼上切开，进行仔细的、创伤性圆环软组织解剖，然后从侧部表面开始，在其中部体上露出股骨。使用前额电梯将肌肉从裸露的骨骼中抬离，并在股骨轴周围进行，直到七到十毫升的中央透析器在四面八方的软组织被清除。接下来，将一个针脚放在横向 epicondyl 的级别上，然后将夹具齐平放置到横向的端骨上，然后插入一根一毫米螺纹尖端 Kirschner 线。接下来，保持夹具在骨骼上的位置，根据夹具孔确定最近的针脚将进入骨骼的位置。根据需要小心地与臀部肌腱的纤维平行，在组织内形成一个小间隙，使近位针通过，从而最大限度地减少肌腱的肌酸损伤。将一毫米非螺纹 k 线钻入间隙，并将钻入的销的高度与松动的销进行比较，以确认该针接合两根皮质。保持夹具与骨骼接触的位置，并钻两根一毫米螺纹 k 线，一根位于未来缺陷站点的两侧，确保针脚接合两根皮质，并放置外部固定杆水平比皮肤高一厘米。拧紧，将杆锁定到位，并夹住多余的销长度。将一个小弯曲的缩回器放在前股骨和后股骨周围，以保护周围的软组织、肌肉和神经血管束，并使用大约五毫米的下垂振荡锯刀片，以及轻，甚至压力，非常仔细地通过中视体创建五毫米的分段缺陷。制造缺陷后，用10毫升0.9%的正常盐水冲洗伤口，并施用肾上腺素给伤口施用0.1毫升0.25%的白霉素，作为镇痛剂和血管炎。将骨形态遗传蛋白-2浸泡海绵支架插入缺陷中，并在简单的中断图案中使用4-0可吸收的缝合线关闭肌肉平面。然后使用 4-0 可吸收的缝合线在运行下切模式关闭皮肤层，然后皮肤胶合以关闭突出针周围的任何间隙，并立即获得股骨的后前放射图像，手术后 4 周和 12 周。仅包含胶原蛋白海绵的负控制缺陷没有证据在近角和近骨边缘之间进行过渡性骨质生成。少量的新的骨骼重塑可以看到直接相邻的切股边缘，而缺陷本身显示缺乏骨质材料，软骨的存在，和一些残余的血肿。含有骨形态遗传蛋白-2浸泡海绵的缺陷在手术后四周就显示出显著的骨愈合，这从跨越缺陷的放射性无情的桥接就证明了这一点。到12周，新的矿物沉积已经在整个缺陷中形成。从切口股骨边缘的无情延伸中可以观察到大量新的近身骨，而编织和跛骨的尖刺在整个缺陷中已经发育。此外，还未看到软骨沉积。在去除外部固定器后，通过 In Vivo 成像系统进行成像，可对缺陷内的生物发光转染细胞进行可视化。例如，在转染后，在三叶细胞中发光，与高西亚荧光素酶的复合mRNA编码，并可用于测量在愈合过程中感兴趣的基因或蛋白质表达的生物变化。销放置对于确保稳定、持久的外部固定非常重要。针脚应直接垂直于股骨，并相互平行，以尽量减少断裂和感染。使用此模型，可以监测生物机制，如特定的蛋白质表达，以更好地了解愈合，不同的脚手架生物材料或疗法也可以测试治疗效果。我们希望这项技术将使未来的研究人员能够更高效地研究骨科愈合与外部固定，以便他们的发现可以转化为改善临床成功。避免吸入麻醉气体，在处理甲醛和 EDTA 时穿戴适当的个人防护设备，在处理手术器械时小心，尤其是钻头和摆动骨锯。

Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

定制设计的基于激光的加热装置顺铂触发释放的热敏脂质体与磁共振成像制导

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position

方法和股骨骨折测试在横盘整理的秋天在臀部位置检测的夹具

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

大鼠牵张成骨的高效可再生性协议

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

一种三肽稳定的油酸纳米乳液

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...