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。

这里所描述的所有方法都已获得南京大学机构动物护理和使用委员会 (IACUC) 的批准。
1. CoCrMo 颗粒制剂
<ol><li>使用制造的高真空三电极直流电流4获得 CoCrMo 粒子。将 CoCrMo 合金置于仪器下 10-3 Pa 真空、0.04 兆帕氩气和氢 3:2 (v/v) 和650阴极电流。</li>
	<li>测量 CoCrMo 颗粒的直径。<ol>
<li>将1毫克的 CoCrMo 颗粒加入1.5 毫升无水乙醇中。</li>
		<li>并用重悬 CoCrMo 颗粒在无水乙醇中的超声震动为28赫, 600 W 为5分钟。</li>
		<li>在透射电子显微镜 (TEM) 的目标表上应用一滴 (约20µL)。捕捉 200 kV 加速度电压和 0.24 nm 分辨率的 TEM 照片系列。</li>
		<li>利用所提供的软件计算 TEM 显微照片中的平均直径和粒度分布。</li>
	</ol>
</li>
	<li>净化内毒素<ol>
<li>在121摄氏度和 15 psi 的15分钟内, 蒸压釜50克颗粒。</li>
		<li>用定量鲎鲎裂解 (拉尔) 法检测内毒素 (< 0.25% 欧盟/毫升被认为是指内毒素缺失)5。</li>
	</ol>
</li>
	<li>并用重悬在磷酸盐缓冲盐水 (PBS) 中的颗粒浓度为50毫克/毫升作为库存解决方案6。</li>
</ol>2. 颅骨骨溶解模型的构建
<ol><li>麻醉6周老 C57BL/J6 小鼠 (每组六只小鼠) withpentobarbital (50 毫克/千克)。使用捏试验评估麻醉的水平。防止眼睛干燥正常生理盐水。</li>
	<li>把老鼠放在俯卧的位置。用剃须刀将颅骨上的毛皮去掉, 用含有75% 乙醇的医用棉球对皮肤进行消毒。</li>
	<li>对于点定位, 分别识别两个点, 包括两个眼睛和耳朵之间的中点。然后, 确定上述两点之间的线, 并用剪刀切割上面线上的皮肤 (图 1A)。</li>
	<li>用手术刀 (图 1B)6从颅骨中取出颅骨骨膜。</li>
	<li>缝合皮肤两端有简单的中断缝合。</li>
	<li>在切口中间做缝合线, 不打结。保持缝合线两端。</li>
	<li>在 calvarias (图 1C)2中嵌入50µL CoCrMo 粒子悬浮 (PBS 中的50毫克/毫升)。</li>
	<li>结在简单中断缝合内的最后一针 (图 1D)。</li>
	<li>再维持2周的老鼠。</li>
</ol>3. 显微 CT 扫描对颅骨骨溶解模型的评价
<ol><li>用二氧化碳牺牲老鼠。斩首在水平平面上的老鼠。取出里面的脑组织, 以及外面的皮肤和皮毛。为进一步的实验收获 calvarias。</li>
	<li>用镊子轻轻地清除颅骨上的软组织。修复清除的 calvarias 在4% 多聚甲醛在4摄氏度24小时, 在微 CT 扫描前浸入 PBS 24 小时的 calvarias。</li>
	<li>用高分辨率微 CT 分析小鼠 calvarias 45 kV 和 550 mA 的18µm 和 X 射线能量设置的等距分辨率。</li>
	<li>采用软件对微 CT 数据进行三维重构。</li>
	<li>定性和定量分析。<ol>
<li>首先, 选择中线缝合周围的正方形区域作为感兴趣区域。</li>
		<li>其次, 测量骨密度 (BMD)、骨容积/总容积 (BV/电视)、小梁数 (tb)、小梁厚度 (tb)、小梁分离/间隔 (tb)、总孔隙率与提供的微 CT 软件的比例。</li>
		<li>第三, 用单向方差分析法对三组进行各种测量。对于差异后的特殊分析, 请应用 Bonferroni 方法2。</li>
	</ol>
</li>
</ol>4. 用 H & E 染色法评价颅骨骨溶解模型
<ol><li>除去石灰质颅骨样品在15% 乙烯二胺四乙酸四酸 (EDTA)-PBS 在4°c。每天更换脱钙液3周。</li>
	<li>将脱钙样品在石蜡中嵌入2厘米 x 1 厘米 x 1 厘米立方, 并将其切成2µm 切片, 在颗粒沉积区内。</li>
	<li>用苏木精和伊红染色切片, 如前所述的7。</li>
	<li>用光镜捕捉整体病理的显微照片。</li>
</ol>

<ol>
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X., Zhang, Z. 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作者没有什么可透露的。

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?...

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。

<table><tbody><tr><td>CoCrMo alloy from prosthesis</td><td>Waldemar Link GmbH &amp;amp; Co</td><td>GEMINI MK II</td><td>Raw material to obtain CoCrMo nanoparticles</td></tr><tr><td>Fabricated high-vacuum three-electrode direct current</td><td>College of Materials Science &amp;amp; Engineering , Nanjing University of Technology</td><td></td><td>Self designed machine</td></tr><tr><td>6 week old male C57BL/6J mice</td><td>Model animal research center of Nanjing University</td><td>N000013</td><td></td></tr><tr><td>100% Ethanol</td><td>Nanjing Reagent</td><td>C0691514023</td><td>Solvent of CoCrMo nanoparticles for transmission electron microscope scanning</td></tr><tr><td>1.5 ml Microcentrifuge tubes</td><td>Taizhou Weierkang Medical Supplies co., LTD</td><td>W603</td><td></td></tr><tr><td>Microanalytical balance</td><td>Shenzhen Qun long Instrument Equipment Co,. LTD</td><td>EX125DZH</td><td></td></tr><tr><td>Ultrasonic shaker</td><td>Shanghai Yuhao scientific instrument co., LTD</td><td>YH-200DH</td><td>To suspend CoCrMo nanoparticles</td></tr><tr><td>Transmission Electron Microscope</td><td>FEI</td><td>Tecnai G20</td><td></td></tr><tr><td>SimplePCI software</td><td>Compix Inc.</td><td>6.6 version</td><td>To calculate the mean diameter and particle size distribution.</td></tr><tr><td>High-handed sterilization pan</td><td>QIULONGYIQI</td><td>KYQL-100DS</td><td>To decontaminate endotoxin</td></tr><tr><td>Limulus Amebocyte Lysate (LAL) Assay</td><td>Charles River</td><td>R13025</td><td>To detect endotoxin&amp;nbsp;</td></tr><tr><td>15 ml Microcentrifuge tubes</td><td>Taizhou Suyi Medical</td><td>B122</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>Boster Biological Technology</td><td>AR0030</td><td>Solvent of CoCrMo nanoparticles stock solution</td></tr><tr><td>Pentobarbital Sodium</td><td>Sigma</td><td>P3761</td><td>To anesthetize mice</td></tr><tr><td>Normal saline</td><td>SACKLER</td><td>SR8572EP-15</td><td>To prevent drying of mice eyes</td></tr><tr><td>75% Ethanol</td><td>Nanjing Reagent</td><td>C0691560275</td><td>Disinfection</td></tr><tr><td>Medical cotton ball</td><td>Shuitao</td><td>1278298933</td><td>Disinfection</td></tr><tr><td>Shaver</td><td>Kemei</td><td>KM-3018</td><td>To shave the fur</td></tr><tr><td>Scissor</td><td>RWD LIFE SCIENCE</td><td>S12005-10</td><td>To incise skin</td></tr><tr><td>Suture</td><td>RWD LIFE SCIENCE</td><td>F34001-01</td><td>To suture skin</td></tr><tr><td>Needle holder</td><td>RWD LIFE SCIENCE</td><td>F33001-01</td><td>To suture skin</td></tr><tr><td>Needle</td><td>RWD LIFE SCIENCE</td><td>R14003-12</td><td>To suture skin</td></tr><tr><td>Vessel forceps</td><td>RWD LIFE SCIENCE</td><td>F22003-09</td><td>To suture skin</td></tr><tr><td>Scalpel</td><td>RWD LIFE SCIENCE</td><td>S31010-01</td><td>To harvest calvaria</td></tr><tr><td>Tweezers</td><td>RWD LIFE SCIENCE</td><td>F12006-10</td><td>To harvest calvaria</td></tr><tr><td>100 &amp;micro;L pipettes</td><td>Eppendorf</td><td>3120000240</td><td>To embed particles suspension in the calvatias</td></tr><tr><td>100 &amp;micro;L pipette tips</td><td>AXYGEN</td><td>T-200-Y</td><td>To embed particles suspension in the calvatias</td></tr><tr><td>5 ml Microtubes</td><td>Taizhou Weierkang Medical Supplies co., LTD</td><td>W621</td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>Servicebio</td><td>G1101</td><td>Fixation</td></tr><tr><td>Micro Computed Tomography&amp;nbsp;</td><td>SkyScan</td><td>SkyScan1176</td><td></td></tr><tr><td>Ethylene Diamine Tetraacetic Acid</td><td>Servicebio</td><td>G1105</td><td>Decalcification</td></tr><tr><td>Paraffin</td><td>Servicebio</td><td>#0001</td><td></td></tr><tr><td>Paraffin slicing machine</td><td>Leica</td><td>RM2125RTS</td><td></td></tr><tr><td>Glass slide</td><td>Servicebio</td><td>G6004</td><td></td></tr><tr><td>Cover glass</td><td>Servicebio</td><td>200</td><td></td></tr><tr><td>HE staining kit</td><td>Servicebio</td><td>#1-5</td><td>HE staining</td></tr><tr><td>Light microscope</td><td>Nikon</td><td>E200</td><td></td></tr></tbody></table>

construction and evaluation of a murine calvarial osteolysis model by exposure to cocrmo particles in aseptic loosening

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。

内部生产的纳米 CoCrMo 粒子的直径约为 50 nm (标准误差 3.56), 由 TEM (图 2) 进行量化。在小鼠 calvarias 接触 CoCrMo 颗粒后, 动物 (每组 n=6) 维持了两周。两周内, 颅骨切口完全愈合, 缝合可能下降。任何局部感染或不愈合都可能影响骨丢失评估。在小鼠牺牲之后, 颅骨样品被收获。然后, 所有的软组织被轻轻地清除, 微 CT 用于量化骨丢失。从三维重建图像和?...

Watch this Scientific Journal Video about Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening at JoVE.com

Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening

CoCrMo 颗粒暴露的小鼠颅骨骨溶解模型的构建与评价

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。

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long ...

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Medicine

7.8K 次观看.  Nanjing University. 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。

视频: Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

利用卡尔瓦里亚共同培养和教条法测量骨重塑和再造肿瘤-骨微环境

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

科研

癌症研究

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

发育生物学

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

多显微镜下的血管生成术在基因修饰的 3 d-PLGA/nHAp 支架上进行颅骨临界骨缺损修复

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

生物工程

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

一个简单的临界尺寸股骨缺损模型小鼠

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

医学

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

在小鼠节段性股临界尺寸缺陷模型的建立接骨板稳定

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

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

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

标准化生物力学条件下小鼠软骨骨折愈合的微创模型分析

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

开发Wistar白化大鼠股骨截骨模型的协议

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

大鼠股骨盒的建立和评估:空腔缺陷模型

Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which involves the application of cells, scaffolds, and cytokines, is considered a promising solution for bone regeneration. Consequently, various animal models that simulate bone defects play a crucial role in exploring the therapeutic potential of tissue engineering for bone healing. In this study, we established a box-shaped cortical bone defect model in the mid-femur of rats, which could serve as an ideal model for assessing the function of biomaterials in promoting bone healing. This box-shaped cortical bone defect was drilled using an oral low-speed handpiece and shaped by a lathe needle. Post-operative micro-CT analysis was immediately conducted to confirm the successful establishment of the box-cavity cortical bone defect. The femurs on the operated side of the rats were then harvested at multiple time points post-surgery (0 days, 2 weeks, 4 weeks, and 6 weeks). The healing process of each sample&#39;s defect area was evaluated using micro-CT, hematoxylin and eosin (H&#38;E) staining, and Masson trichrome staining. These results demonstrated a healing pattern consistent with intramembranous ossification, with healing essentially complete by 6 weeks. The categorization of this animal model&#39;s healing process provides an effective in vivo method for investigating novel biomaterials and drugs that target intramembranous ossification during bone tissue defect healing.

作者聚焦:用于增强生物材料和骨再生评估的箱腔皮质方法

Here, we present a protocol for creating a box-cavity defect in rat femoral diaphysis tissue. This model can assess biomaterial performance under biomechanical stress and explore mechanisms of bone regeneration related to intramembranous osteogenesis.

在大鼠股骨皮质骨中创建箱腔缺损模型

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which ...

CoCrMo 颗粒暴露的小鼠颅骨骨溶解模型的构建与评价

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此视频中的章节

利用卡尔瓦里亚共同培养和教条法测量骨重塑和再造肿瘤-骨微环境

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

多显微镜下的血管生成术在基因修饰的 3 d-PLGA/nHAp 支架上进行颅骨临界骨缺损修复

一个简单的临界尺寸股骨缺损模型小鼠

在小鼠节段性股临界尺寸缺陷模型的建立接骨板稳定

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

标准化生物力学条件下小鼠软骨骨折愈合的微创模型分析

开发Wistar白化大鼠股骨截骨模型的协议

在大鼠股骨皮质骨中创建箱腔缺损模型

在大鼠股骨皮质骨中创建箱腔缺损模型