CoCrMo 颗粒暴露的小鼠颅骨骨溶解模型的构建与评价
Summary
这篇手稿描述了一个小鼠颅骨骨溶解模型的接触 CoCrMo 颗粒, 这构成了一个理想的动物模型, 以评估磨损颗粒和各种细胞之间的相互作用的无菌松动。
Abstract
磨粒引起的骨溶解是关节置换术失败无菌性松动的主要原因, 但其基本机制尚不清楚。由于检测和零星发生所需的长期随访, 对临床病例 ofparticle 性骨溶解的发病机制进行评估具有挑战性。因此, 需要进行进一步研究的最佳动物模型。通过接触 CoCrMo 颗粒建立的小鼠颅骨骨溶解模型, 是评价无菌松动中颗粒与各种细胞相互作用的有效和有效的工具。在该模型中, CoCrMo 粒子首先获得高真空三电极直流和悬浮在磷酸盐缓冲盐水浓度为50毫克/毫升。然后, 50 µL 的结果被应用到小鼠颅骨的中间后, 通过尖锐解剖分离颅骨骨膜。两周后, 小鼠被献祭, 颅骨标本被收割;定性和定量评价是由苏木精和伊红染色和微计算机断层扫描进行的。该模型的优点包括程序简单性、骨质丢失定量评价、骨溶解快速发展、潜在使用转基因或挖空模型以及相对较低的成本。然而, 这种模型不能用来评估机械力和颗粒在无菌松动的慢性影响。暴露于 CoCrMo 颗粒的小鼠颅骨骨溶解模型是评估磨损颗粒与各种细胞之间相互作用的理想工具, 例如, 在无菌性松动中,如:、细胞、成骨细胞、细胞和破骨。
Introduction
无菌松动是全髋关节置换术 (临区) 和全膝关节置换术 (TKA) 失败的最常见原因, 需要修订手术1。但是, 基础机制仍然不清楚2。需要长时间的随访, 以检测颗粒诱导的骨溶解, 其发生是罕见的;因此, 探讨其在临床病例中的发病机制具有挑战性。因此, 对复杂的细胞和组织机制的进一步研究需要体内实验中的磨损颗粒诱导的骨溶解模型和体外检测与骨骼稳态相关的细胞3。有效的动物模型对于揭示磨损颗粒对骨丢失的影响是很重要的, 为进一步的细胞化验提供了证据。
由接触 CoCrMo 颗粒构建的小鼠颅骨骨溶解模型是一种有效、有效的评估无菌松动中颗粒与不同细胞间相互作用的方法。在该模型中, CoCrMo 颗粒通过诱导巨噬细胞炎性细胞因子, 活化破骨细胞, 抑制成骨生长, 促进成骨细胞凋亡, 引起颅骨溶解。
建立这个模型只需要两个星期的时间。骨溶解可以可视化和量化的苏木精和伊红 (H & E) 染色和微计算机断层扫描 (微 CT)2。此外, 该模型的成本相对较低, 转基因和挖空鼠标模型可以用来筛选大量的化合物在不同剂量的3。
建立和评估这个模型的程序是简单的。首先, 用高真空三电极直流电流和悬浮在磷酸盐缓冲盐水 (PBS) 中获得了 CoCrMo 颗粒, 浓度为50毫克/毫升。然后, 50 µL 的结果被应用到小鼠颅骨的中间后, 通过尖锐解剖分离颅骨骨膜。两周后, 小鼠被献祭, 颅骨标本被收割;采用 H & E 染色 andmicro CT 进行定性和定量分析。
通过接触 CoCrMo 颗粒构建的小鼠颅骨骨溶解模型, 是评估 CoCrMo 颗粒与各种细胞之间相互作用的理想工具, 如巨细胞、成纤维体、成骨细胞和破骨动物, 无菌松动。
Protocol
Representative Results
Discussion
在小鼠体内, 有两种主要的颗粒诱导骨溶解方法: 气袋模型和颅骨骨溶解模型。在气袋模型中, 首先建立一个皮下生成的气囊, 然后将粒子引入和植入到骨骼组织中8。囊壁模仿骨膜在无菌松动。然而, 骨移植 nonvascular 没有生物活性, 这使得很难评估颗粒和骨组织之间的直接相互作用。颅骨骨溶解模型在气袋对应物上有几个优点。首先, 磨损颗粒直接暴露于颅骨, 使其能够评估磨损颗?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究获得中国国家自然科学基金 (81572111)、江苏省临床科技项目基金会 (BL2012002)、南京市科研项目 (201402007)、自然科学的支持。江苏省基础 (BK20161385), 中国医生协会 (2015COS0810) 特别基金会。
Materials
| CoCrMo alloy from prosthesis | Waldemar Link GmbH & Co | GEMINI MK II | Raw material to obtain CoCrMo nanoparticles |
| Fabricated high-vacuum three-electrode direct current | College of Materials Science & Engineering , Nanjing University of Technology | Self designed machine | |
| 6 week old male C57BL/6J mice | Model animal research center of Nanjing University | N000013 | |
| 100% Ethanol | Nanjing Reagent | C0691514023 | Solvent of CoCrMo nanoparticles for transmission electron microscope scanning |
| 1.5 ml Microcentrifuge tubes | Taizhou Weierkang Medical Supplies co., LTD | W603 | |
| Microanalytical balance | Shenzhen Qun long Instrument Equipment Co,. LTD | EX125DZH | |
| Ultrasonic shaker | Shanghai Yuhao scientific instrument co., LTD | YH-200DH | To suspend CoCrMo nanoparticles |
| Transmission Electron Microscope | FEI | Tecnai G20 | |
| SimplePCI software | Compix Inc. | 6.6 version | To calculate the mean diameter and particle size distribution. |
| High-handed sterilization pan | QIULONGYIQI | KYQL-100DS | To decontaminate endotoxin |
| Limulus Amebocyte Lysate (LAL) Assay | Charles River | R13025 | To detect endotoxin |
| 15 ml Microcentrifuge tubes | Taizhou Suyi Medical | B122 | |
| Phosphate-buffered saline | Boster Biological Technology | AR0030 | Solvent of CoCrMo nanoparticles stock solution |
| Pentobarbital Sodium | Sigma | P3761 | To anesthetize mice |
| Normal saline | SACKLER | SR8572EP-15 | To prevent drying of mice eyes |
| 75% Ethanol | Nanjing Reagent | C0691560275 | Disinfection |
| Medical cotton ball | Shuitao | 1278298933 | Disinfection |
| Shaver | Kemei | KM-3018 | To shave the fur |
| Scissor | RWD LIFE SCIENCE | S12005-10 | To incise skin |
| Suture | RWD LIFE SCIENCE | F34001-01 | To suture skin |
| Needle holder | RWD LIFE SCIENCE | F33001-01 | To suture skin |
| Needle | RWD LIFE SCIENCE | R14003-12 | To suture skin |
| Vessel forceps | RWD LIFE SCIENCE | F22003-09 | To suture skin |
| Scalpel | RWD LIFE SCIENCE | S31010-01 | To harvest calvaria |
| Tweezers | RWD LIFE SCIENCE | F12006-10 | To harvest calvaria |
| 100 µL pipettes | Eppendorf | 3120000240 | To embed particles suspension in the calvatias |
| 100 µL pipette tips | AXYGEN | T-200-Y | To embed particles suspension in the calvatias |
| 5 ml Microtubes | Taizhou Weierkang Medical Supplies co., LTD | W621 | |
| 4% Paraformaldehyde | Servicebio | G1101 | Fixation |
| Micro Computed Tomography | SkyScan | SkyScan1176 | |
| Ethylene Diamine Tetraacetic Acid | Servicebio | G1105 | Decalcification |
| Paraffin | Servicebio | #0001 | |
| Paraffin slicing machine | Leica | RM2125RTS | |
| Glass slide | Servicebio | G6004 | |
| Cover glass | Servicebio | 200 | |
| HE staining kit | Servicebio | #1-5 | HE staining |
| Light microscope | Nikon | E200 |
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