This work was supported by the Sichuan Provincial Administration of Traditional Chinese Medicine (2021YJ0175) and the Graduate Research Innovation Project of the School of Clinical Medicine (LCYJSKT2023-11), Chengdu University of Traditional Chinese Medicine.

The animal protocol has been approved by the Animal Ethical Committee of Chengdu University of Traditional Chinese Medicine (Record number: 2020-34). Female C57BL/6J mice (12-week old, n = 14) were divided into two groups randomly, a sham-operated group (Sham group, n = 7) and a model group (OVX group, n = 7). Animals were purchased from a commercial supplier (see Table of Materials). All mice were kept in individual cages at 22-26 &#176;C with 45%-55% humidity, allowed to adapt to their new environment for 1 week, and provided free access to water and diet. All animal experimental studies were conducted at Chengdu University of Traditional Chinese Medicine, and all efforts were made to minimize animal suffering.
1. Animal model preparation
<ol>
	<li>Anesthetize the 12-week-old mouse by intraperitoneal injection with 1.25% Avertin (Tribromoethanol in tert-amyl alcohol, see Table of Materials) at a dosage of 0.02 mL/g. Position the mouse prone on a sterile surgical operating table, and immobilize its limbs by taping them securely.</li>
	<li>Use scissors (see Table of Materials) to trim any hair that may affect the surgical operation. 
	NOTE: It is recommended to avoid skin damage while preparing the surgical site.</li>
	<li>Wash hands thoroughly and put on surgical gloves. Disinfect the mouse&#39;s back three times with povidone-iodine (see Table of Materials), and use medical gauze (see Table of Materials) to dry it off. 
	NOTE: Be cautious; getting the hair too wet during disinfection may lead to postoperative hypothermia in mice.</li>
	<li>Make an incision approximately 0.5-1.0 cm long, located 1 cm away from one-third of the midline of the mouse&#39;s back, using a scalpel (see Table of Materials). Gently separate the fascia and cut through the muscle with tissue scissors (see Table of Materials) until the ovary is visible. Ligate the surrounding blood vessels with non-absorbable sutures (see Table of Materials) and remove the ovary.</li>
	<li>Rinse the cavity using 0.9% saline solution, then suture the skin, muscle, and fascia separately (see Table of Materials).</li>
	<li>Repeat the same sequence of steps on the other side.</li>
	<li>Remove peri-ovarian fat of the same size as the ovary, and perform the same surgery procedure mentioned above for the remaining steps to establish the sham-operated model.</li>
	<li>Allow a recovery period of 1 week after the surgery. After 8 weeks, osteoporotic mouse models will be successfully established9,10.</li>
</ol>
2. Micro-CT scanning
<ol>
	<li>Euthanize the mouse by excess CO2 inhalation. Remove as much soft tissue as possible from the mouse femur after euthanasia and obtain a fresh sample for scanning. 
	NOTE: While there is no perfect solution for sample preservation, certain steps can be taken to maximize the quality of Micro-CT scan results. Before fixation, take care to remove as much surrounding tissue as possible. Formalin or buffered formalin is the most preferred method of fixation with storage in PBS11,12.</li>
	<li>Double-click the micro-CT imaging system software icon on the desktop to start the system (see Table of Materials). Select a sample bed compatible with the sample field of view (FOV) of 18 mm x 18 mm. Load the appropriate sample bed into the machine and close the hatch.</li>
	<li>Click the Warm-up button in the Control Panel. 
	NOTE: A short warm-up time was required. Do not attempt to open the hatch when X-rays are being generated. Check the X-Ray On light on the front panel and computer monitor to confirm if X-rays are being generated.</li>
	<li>Click the Menu button to set a new database, then create a new sample and study.</li>
	<li>Select Manual from the Menu drop-down list and enter custom voltage and current values in the Control Panel. Set Voltage (kV) to 90, Current (&#181;A) to 80, Scan Mode to High resolution, 14 min, and FOV (mm) to 18 mm x 18 mm. 
	NOTE: Refer to the manual for different micro CT systems to set the appropriate parameters.</li>
	<li>Position the bone securely with plastic film in the sample bed. Close the hatch. Ensure that the subject is centered precisely in the X-capture window by pressing the adjustment buttons on the machine. 
	NOTE: Adjusting the sample must be slow and gentle to avoid it falling into the machine by accident.</li>
	<li>Click the Start button to start the CT scan.</li>
</ol>
3. CT data analysis
<ol>
	<li>Enter the software (see Table of Materials), and select the data to be analyzed. Click Sub and set Pixel Size to 10 &#181;m. Move and resize the region of interest (ROI) to include the distal femur above the growth plate. Click on Start (see Figure 1). 
	NOTE: The subvolume reconstruction pixel size was selected at 10 &#181;m since the thickness of the imaging trabeculae was estimated to be about 20-30 &#181;m based on histological data from previous studies13. If the signal-to-noise ratio is insufficient for data processing, a high-resolution 1 h scan must be performed.</li>
	<li>Click the Analysis 3D button to get the resulting 3D reconstruction.</li>
	<li>Export the data from the micro-CT system to the computer for analysis.</li>
	<li>Import the reconstructed CT data. Click Process &#62; Image Calculator, then click Region Pad &#62; Interactive. Adjust the yellow checkbox to the appropriate ROI. 
	NOTE: The region of interest (ROI) begins approximately 540 &#181;m proximal to the growth plate and extends 1600 &#181;m proximally to assess the actual bone metabolism and remodeling.</li>
	<li>Select the Bone Microarchitecture Analysis (BMA) add-on (see Table of Materials). Click Segment Cortex and then Segment Trabeculae. 
	NOTE: Both trabecular and cortical boneare automatically selected. Normally no manual adjustment is required.</li>
	<li>Click on Save Final Object Map and then click Measure Bone to calculate bone morphometric indices. 
	&#8203;NOTE: Only relative bone mineral density (BMD) was calculated here since no control was added.</li>
</ol>
4. Decalcification of bone tissue
<ol>
	<li>Fix the bone specimens in 4% paraformaldehyde (see Table of Materials) for 24 h. Wash the specimens thrice with PBS (see Table of Materials) for 20 min each time.</li>
	<li>Rinse the tissue thrice with distilled water for 20 min each time. Transfer the tissue to a decalcification solution containing EDTA (see Table of Materials) and decalcify for 30 days with a weekly solution change until endpoint. 
	NOTE: Needle pricking, hand pinching, and clamping are used to terminate decalcification when the bone tissue becomes soft, or there is no sense of resistance when needling. The physical method of detection can cause some damage to the tissue structure, so try to avoid excessive force or repeated testing.</li>
	<li>Take the tissue out of the fixative and use a scalpel to trim the tissue in a fume hood. Put the trimmed tissue and the corresponding label in a dehydration box.</li>
	<li>Put the dehydration box in a basket and dehydrate it in a tissue processor (see Table of Materials) with gradient alcohol (75% ethanol for 4 h, 85% ethanol for 2 h, 90% ethanol for 2 h, 95% ethanol for 1 h, anhydrous ethanol for 30 min, anhydrous ethanol II for 30 min, xylene I for 5-10 min, xylene II for 5-10 min, wax I for 1 h, wax II for 1 h, wax III for 1 h).</li>
	<li>Use an embedding machine (see Table of Materials) to embed the wax-soaked tissue. Pour the melted wax into an embedding box and place the tissue from the dehydration box before the wax hardens.</li>
	<li>Orient the tissue according to the embedding surface and attach the corresponding label. Cool on a -20 &#176;C freezing table (see Table of Materials). Remove the wax block from the embedding box after solidifying and trim the wax block as needed (see Table of Materials).</li>
	<li>Cut the trimmed wax block into 3 &#181;m thick slides on a microtome (see Table of Materials). Float the slides on 40 &#176;C warm water on a tissue spreading machine (see Table of Materials) to flatten the tissue and scoop them up with a slide.</li>
	<li>Bake in a 60 &#176;C oven (see Table of Materials) until the water evaporates and the wax melts. Take out and store at room temperature (RT) for later use.</li>
</ol>
5. HE staining
<ol>
	<li>Put the slides in xylene I for 20 min, then in xylene II for 20 min. Further, put the slides in anhydrous ethanol I and anhydrous ethanol II for 5 min each, 75% alcohol for 5 min, and then wash the slides with tap water (see Table of Materials).</li>
	<li>Incubate in hematoxylin (see Table of Materials) for 3-5 min. Differentiate the specimens with a hydrochloric acid solution (see Table of Materials). Treat the slides with an ammonia solution (see Table of Materials) for bluing, and then wash the slides with water.</li>
	<li>Put the slides in 85%, 95% gradient alcohol for dehydration, and stain with eosin solution (see Table of Materials) for 5 min.</li>
	<li>Put the slides in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and anhydrous ethanol III for 5 min. Treat the slides with xylene I for 5 min, xylene II for 5 min, and mount with neutral balsam (see Table of Materials).</li>
	<li>Examine each slide under the microscope. Then, choose the representative slices for panoramic scanning (see Table of Materials). 
	&#8203;NOTE: At least three consecutive slices are required for each sample. Before panoramic scanning, the slices must be examined under a microscope to ensure that they are complete and clear. Ensure that the bone marrow cavity, cortical bone, and cancellous bone are fully displayed and that different types of bone cells can be seen.</li>
</ol>
6. HE image analysis
<ol>
	<li>Open HE images with CaseViewer software (see Table of Materials). Select the region of interest (ROI) on the slide, and save it as a color image. 
	NOTE: The selection of ROI is consistent with the above-mentioned software.</li>
	<li>Open the image in ImageJ (see Table of Materials). Select the Wand tool from the toolbar. Click on the trabecular bone.</li>
	<li>Adjust the tolerance and contiguous settings as needed. Go to Image &#62; Adjustments &#62; Black &#38; White and click OK. Repeat the steps for other areas of the bone.</li>
	<li>Invert the selection and fill it with white color. Save the image as a mask for analysis (see&#160;Figure 2). 
	NOTE: For the detailed methodology, refer to the previously published work14.</li>
	<li>Open the mask in the analysis software15,16 (see Table of Materials). Run Process &#62; Binary &#62; Make Binary to convert the color image to a binary image.</li>
	<li>Use plugin17 (see Table of Materials) in the software to analyze structural parameters: Run Area/Volume Fraction to calculate bone volume to total volume of bone (BV/TV [%])18,19. 
	NOTE: This method is applicable for bone trabeculae and bone marrow, filling the entire ROI in the HE images.</li>
</ol>

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

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The authors have nothing to disclose.

Osteoporosis can lead to frequent fractures, which are costly, can cause pain, disability, or even death, and seriously affect the quality of life of patients20. Over the years, the ovariectomy model has been recognized as one of the standard methods for studying osteoporosis21. The most common preclinical animal model for osteoporosis is the ovariectomized (OVX) rat. Despite this, the majority of research into the mechanisms of bone disorders, including osteoporosis, has b...

Osteoporosis is a prevalent metabolic bone disease, especially among the elderly, and is associated with an increased risk of fragility fractures. As osteoporosis becomes more common in China1, there will be a growing demand for studying the bone structures of small animals2,3. The previous methods of measuring bone loss rely on the outcomes of two-dimensional dual-energy x-ray absorptiometry. However, this does not capture the changes in the architectural microstructure of the trabecular bone, which is a key factor for skeletal strength4. The microstructure of bone affects its strength, stiffness, and fracture resistance. By comparing bone microarchitecture in normal and pathological states, changes in bone tissue morphology, structure, and function caused by osteoporosis can be identified. This information contributes to the understanding of the development of osteoporosis and its association with other diseases. 
Micro-computed tomography (Micro-CT) imaging has recently become a popular technique for bone morphology assessment, where it can provide accurate and comprehensive data on bone structure and density parameters such as bone volume fraction, thickness, and separation5,6. At the same time, the Micro-CT results can be affected by the analysis software7. Different methods of image acquisition, evaluation, and reporting are used by various commercial Micro-CT systems. This inconsistency makes it hard to compare and interpret the results reported by different studies5. Also, it cannot currently replace bone histomorphometry in providing researchers with information on cellular-level parameters in the skeletal system8. Meanwhile, histologic techniques allow direct observation and measurement of the microscopic morphology of bone. Hematoxylin and eosin (HE) staining is a common staining technique used in histology to visualize the general structure of cells and tissues. It is used to identify the presence of bone tissue and its microarchitecture.
This article uses Micro-CT combined with tissue slicing technique (Hematoxylin-Eosin [HE] staining) to collect bone tissue images and perform quantitative analysis of trabecular bone to evaluate the changes of bone microstructure in an osteoporosis mouse model.

<table><tbody><tr><td>4% Paraformaldehyde</td><td>Biosharp</td><td>BL539A</td><td></td></tr><tr><td>Adobe Photoshop</td><td>Adobe Inc.</td><td></td><td></td></tr><tr><td>Ammonia Solution</td><td>Chengdu Kolon Chemical Co., Ltd</td><td>2021070101</td><td></td></tr><tr><td>Anatomical Forceps</td><td>Jinzhong surgical instrument Co., Ltd</td><td>J3C030</td><td></td></tr><tr><td>Anhydrous Ethanol</td><td>Chengdu Kolon Chemical Co., Ltd</td><td>2022070501</td><td></td></tr><tr><td>Automatic Dyeing Machine</td><td>Thermo scientific</td><td>Varistain&amp;trade; Gemini ES</td><td></td></tr><tr><td>Bone Microarchitecture Analysis Add-on</td><td>AnalyzeDirect, Inc</td><td></td><td></td></tr><tr><td>C57BL/6J mice</td><td>SPF (Beijing) Biotechnology Co., Ltd.</td><td></td><td></td></tr><tr><td>Carrier Slides</td><td>Nantong Mei Wei De Experimental Equipment Co., Ltd</td><td>220518001</td><td></td></tr><tr><td>Coverslips</td><td>Nantong Mei Wei De Experimental Equipment Co.</td><td>220518001</td><td></td></tr><tr><td>Decalcification Solution</td><td>Wuhan Xavier Biotechnology Co., Ltd</td><td>CR2203047</td><td></td></tr><tr><td>Delicate Scissors</td><td>Jinzhong surgical instrument Co., Ltd</td><td>ZJA010</td><td></td></tr><tr><td>Embedding box marking machine</td><td>Thermo scientific</td><td>&amp;nbsp;PrintMate AS</td><td></td></tr><tr><td>Embedding Machine</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JB-P5</td><td></td></tr><tr><td>Fiji: ImageJ</td><td>National Institutes of Health, USA</td><td></td><td></td></tr><tr><td>Film Sealer</td><td>Thermo scientific</td><td>Autostainer 360</td><td></td></tr><tr><td>Freezing Table</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JB-L5</td><td></td></tr><tr><td>H&amp;amp;E Staining Kit</td><td>Leagene</td><td>DH0020</td><td></td></tr><tr><td>Hydrochloric Acid Solution</td><td>Sichuan Xilong Science Co., Ltd</td><td>210608</td><td></td></tr><tr><td>ImageJ2 Plugin</td><td></td><td>BoneJ 7.0.16</td><td></td></tr><tr><td>Medical Gauze</td><td>Shandong Ang Yang Medical Technology Co.</td><td></td><td></td></tr><tr><td>Mersilk 3-0 Silk Braided Non-Absorbable Sutures</td><td>Ethicon, Inc.</td><td>SA84G</td><td></td></tr><tr><td>Needle Holder</td><td>Jinzhong surgical instrument Co., Ltd</td><td>J32010</td><td></td></tr><tr><td>Neutral Balsam</td><td>Sinopharm Group Chemical Reagent Co., Ltd</td><td>10004160</td><td></td></tr><tr><td>Oven</td><td>Shanghai Yiheng Scientific Instruments Co., Ltd</td><td>DHG-9240A</td><td></td></tr><tr><td>PANNORAMIC Digital Slide Scanners</td><td>3DHISTECH Ltd.&amp;nbsp;</td><td>PANNORAMIC DESK/MIDI/250/1000</td><td></td></tr><tr><td>PBS buffer</td><td>Biosharp</td><td>G4202</td><td></td></tr><tr><td>Povidone-iodine solution 5%</td><td>Chengdu Yongan Pharmaceutical Co., Ltd</td><td></td><td></td></tr><tr><td>Quantum GX2 microCT Imaging System</td><td>PerkinElmer, Inc.</td><td></td><td></td></tr><tr><td>Rotary Microtome</td><td>Thermo scientific</td><td>HM325</td><td></td></tr><tr><td>Scalpel</td><td>Quanzhou Excellence Medical Co., Ltd</td><td>20170022</td><td></td></tr><tr><td>Scan &amp;amp; Browse Software</td><td>3DHISTECH Ltd.&amp;nbsp;</td><td>CaseViewer2.4</td><td></td></tr><tr><td>Single-Use Sterile Rubber Surgical Gloves</td><td>Guangdong Huitong Latex Products Group Co., Ltd</td><td>22B141EO</td><td></td></tr><tr><td>Sodium Chloride Solution 0.9%</td><td>Sichuan Kelun Pharmaceutical Co., Ltd</td><td></td><td></td></tr><tr><td>Sterile Hypodermic Syringes for Single Use</td><td>Shandong Weigao Group Medical Polymer Products&amp;nbsp; Co., Ltd</td><td></td><td></td></tr><tr><td>Sterile Medical Suture Needles</td><td>Shanghai Pudong Jinhuan Medical Products Co., Ltd.&amp;nbsp;</td><td>PW8068</td><td></td></tr><tr><td>Tissue Processor</td><td>Thermo scientific</td><td>STP420 ES</td><td></td></tr><tr><td>Tissue Spreading and Baking Machine</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JK-6</td><td></td></tr><tr><td>Tribromoethanol</td><td>Nanjing Aibei Biotechnology Co., Ltd</td><td>M2920</td><td></td></tr><tr><td>Wax Trimmer</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JXL-818</td><td></td></tr><tr><td>Xylene</td><td>Chengdu Kolon Chemical Co., Ltd</td><td>2022051901</td><td></td></tr></tbody></table>

trabecular bone microarchitecture evaluation in an osteoporosis mouse model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Micro-CT analysis 
We measured the trabecular microarchitectural parameters in mice from both groups and reported their mean values and SDs in Table 1. The distribution of some parameters (i.e., the ratio of bone volume to total tissue volume, trabecular thickness, trabecular separation) within each group are illustrated in Figure 3.
These results indicate significant differen...

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Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

trabecular-bone-microarchitecture-evaluation-an-osteoporosis-mouse

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

2.1K Views.  Hospital of Chengdu University of Traditional Chinese Medicine. This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Video: Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

Bioengineering

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different bone loss conditions. However, the standard approach for trabecular analysis of micro-CT images is slice-by-slice semi-automatic hand-contouring, which is labor intensive and error prone. Described here is an efficient method for automatic segmentation of trabecular bones according to the bone's outer boundaries, where trabecular bones can be identified and segmented automatically with accuracy with less operator bias when appropriate segmentation parameters are set. To profile satisfactory segmentation parameters, an image stack of segmentation results is displayed, where all possible combinations of the segmentation parameters are changed one by one in sequence, and segmentation results with associated parameters can easily be visually checked. As a quality-control feature of the plugin, simulated standard objects are quantified where the measured quantities can be compared with theoretical values. Layer-by-layer quantification of trabecular properties and trabecular thicknesses are reported by such a plugin, and the distributions of such properties within the selected regions can be profiled easily. Although layer-by-layer quantification retains more information about trabecular bones and facilitates further statistical analysis of structural changes, such measures are unavailable from the output of current commercial software, where only a single quantified value for each parameter is reported for each sample. Therefore, the described workflows are better approaches for analyzing trabecular bones with accuracy and efficiency.

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone&#39;s outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial software.

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

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

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long follow-ups necessary for detection and sporadic occurrence, it is challenging to assess the pathogenesis ofparticle-induced osteolysis in clinical cases. Hence, optimal animal models are required for further studies. The murine model of calvarial osteolysis established by exposure to CoCrMo particles is an effective and valid tool for assessing the interactions between particles and various cells in aseptic loosening. In this model, CoCrMo particles were first obtained by high-vacuum three-electrode direct current and resuspended in phosphate-buffered saline at a concentration of 50 mg/mL. Then, 50 &#181;L of the resulting suspension was applied to the middle of the murine calvaria after separation of the cranial periosteum by sharp dissection. After two weeks, the mice were sacrificed, and calvaria specimens were harvested; qualitative and quantitative evaluations were performed by hematoxylin and eosin staining and micro computed tomography. The strengths of this model include procedure simplicity, quantitative evaluation of bone loss, rapidity of osteolysis development, potential use transgenic or knockout models, and a relatively low cost. However, this model cannot to be used to assess the mechanical force and chronic effects of particles in aseptic loosening. Murine calvarial osteolysis model generated by exposure to CoCrMo particles is an ideal tool for assessing the interactions between wear particles and various cells, e.g., macrophages, fibroblasts, osteoblasts and osteoclasts, in aseptic loosening.

This manuscript describes a murine calvarial osteolysis model by exposure to CoCrMo particles, which constitutes an ideal animal model for assessing the interactions between wear particles and various cells in aseptic loosening.

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

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley (SD) rats were randomly divided into six groups: a sham-operated group, a model group (OVX), a progynova group, and BZBS groups (1, 2, and 4 d/kg/d). An ovariectomy was performed on all rats except those in the sham-operated group. Micro-computed tomography (micro-CT) scanning, hematoxylin and eosin (H&amp;E) staining, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) detection were performed after 4 months of BZBS treatment. As a result, compared with the OVX group, rats treated with BZBS showed an increased number and area of trabecular bone and bone marrow cells, and a decreased number of adipose cells. The bone volume, trabecular number, and trabecular thickness of the right tibia in the medication groups increased and the trabecular space decreased. The 17&#946;-estradiol and serum calcium levels in the medication groups were elevated, but the levels of serum phosphorus, sclerostin, &#946;-CTX, and TRACP-5b were decreased. In the medication groups, the RANKL and sclerostin levels were decreased, while the osteoprotegerin (OPG) level was increased. In conclusion, this protocol systematically evaluated the therapeutic effects and potential molecular mechanisms of Chinese herbal compounds in ovariectomized rats with a variety of techniques.

Presented here is a protocol to show that Bazi Bushen capsule (BZBS) can regulate the RANKL/OPG signaling pathway in the ovariectomized rodent model through its estrogen-like effect.

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley ...

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms and pharmaceutical treatments of osteoporosis. Cathepsin K (Ctsk)-Cre mice have been widely used for functional studies of osteoclasts. The signal transducer and activator of transcription 3 (STAT3) is relevant in bone homeostasis, but its role in osteoclasts in vivo remains poorly defined. To provide the in vivo evidence that STAT3 participates in osteoclast differentiation and bone metabolism, we generated an osteoclast-specific Stat3 deletion mouse model (Stat3 fl/fl; Ctsk-Cre) and analyzed its skeletal phenotype. Micro-CT scanning and 3D reconstruction implied increased bone mass in the conditional knockout mice. H&#38;E staining, calcein and alizarin red double staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed to detect bone metabolism. In short, this protocol describes some canonical methods and techniques to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

This protocol describes a canonical method to understand the critical genes controlling osteoclast activity in vivo. This method uses a transgenic mouse model and some canonical techniques to analyze skeletal phenotype.

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

Biology

Elastic Tape Rat Model of Steroid-Induced Femoral Head Osteonecrosis Enhancement

Tension-Free Weight-Bearing Model of Steroid-Induced Osteonecrosis of Femoral Head in Rats

Unlike humans, rats are animals that walk on all fours, while humans are bipedal animals that stand, and the hips are subjected to tremendous pressure when walking and standing. In rat steroid-induced femoral head necrosis models, the biomechanical characteristics of the human hip under higher pressure often need to be simulated. Some scholars try to emulate the state of human hip pressure by making rats bear a certain weight, but fixing the weight-bearing object on the rat is tough. Rats can easily break free of immobilization, and sticking the weight on the rats with adhesive tape will cause the rats to suffocate or die from intestinal obstruction. Our research group used elastic therapeutic tape to perform tension-free immobilization of weight-bearing objects in rats so that the rats could breathe freely and not break away from the immobilization under weight-bearing conditions. Compared to the usual steroid-induced femoral head necrosis rat model, we found that this weight-bearing intervention can aggravate the progression of femoral head necrosis in rats.

Author Spotlight: Developing a Rat Model for Weight-Bearing Intervention to Investigate Osteonecrosis of the Femoral Head

The protocol shows building a simple and cost-effective rat weight-bearing training model for steroid-induced osteonecrosis of the femoral head using elastic therapeutic tape.

Unlike humans, rats are animals that walk on all fours, while humans are bipedal animals that stand, and the hips are subjected to tremendous pressure ...

3D Visualization of Bone Remodeling Under Tensile Load in Suture Expansion Mice

A 3-D Visualization Technique for Bone Remodeling in a Suture Expansion Mouse Model

Craniofacial sutures play a crucial role beyond being fibrous joints connecting craniofacial bones; they also serve as the primary niche for calvarial and facial bone growth, housing mesenchymal stem cells and osteoprogenitors. As most craniofacial bones develop through intramembranous ossification, the sutures' marginal regions act as initiation points. Due to this significance, these sutures have become intriguing targets in orthopedic therapies like spring-assisted cranial vault expansion, rapid maxillary expansion, and maxillary protraction. Under orthopedic tracing force, suture stem cells are rapidly activated, becoming a dynamic source for bone remodeling during expansion. Despite their importance, the physiological changes during bone remodeling periods remain poorly understood. Traditional sectioning methods, primarily in the sagittal direction, do not capture the comprehensive changes occurring throughout the entire suture. This study established a standard mouse model for sagittal suture expansion. To fully visualize bone remodeling changes post-suture expansion, the PEGASOS tissue clearing method was combined with whole-mount EdU staining and calcium chelating double labeling. This allowed the visualization of highly proliferating cells and new bone formation across the entire calvarial bones following expansion. This protocol offers a standardized suture expansion mouse model and a 3-D visualization method, shedding light on the mechanobiological changes in sutures and bone remodeling under tensile force loading.

Author Spotlight: PEGASOS Tissue Clearing Technique to Visualize Bone Remodeling 

This protocol presents a standardized suture expansion mouse model and a 3-D visualization method to study the mechanobiological changes of the suture and bone remodeling under tensile force loading.

Craniofacial sutures play a crucial role beyond being fibrous joints connecting craniofacial bones; they also serve as the primary niche for calvarial and ...

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

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Rozdziały w tym wideo

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

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

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Tension-Free Weight-Bearing Model of Steroid-Induced Osteonecrosis of Femoral Head in Rats

A 3-D Visualization Technique for Bone Remodeling in a Suture Expansion Mouse Model