Name: Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model
Uploaded: 2023-09-08T14:10:00
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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 ...

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

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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.

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			<td>Adobe Photoshop</td>
			<td>Adobe Inc.</td>
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			<td>Chengdu Kolon Chemical Co., Ltd</td>
			<td>2021070101</td>
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			<td>Analyze 12.0</td>
			<td>AnalyzeDirect, Inc</td>
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			<td>Anatomical Forceps</td>
			<td>Jinzhong surgical instrument Co., Ltd</td>
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			<td>Chengdu Kolon Chemical Co., Ltd</td>
			<td>2022070501</td>
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			<td>Thermo scientific</td>
			<td>Varistain&#8482; Gemini ES</td>
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			<td>Bone Microarchitecture Analysis Add-on</td>
			<td>AnalyzeDirect, Inc</td>
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			<td>C57BL/6J mice</td>
			<td>SPF (Beijing) Biotechnology Co., Ltd.</td>
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			<td>Carrier Slides</td>
			<td>Nantong Mei Wei De Experimental Equipment Co., Ltd</td>
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			<td>Coverslips</td>
			<td>Nantong Mei Wei De Experimental Equipment Co.</td>
			<td>220518001</td>
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			<td>Decalcification Solution</td>
			<td>Wuhan Xavier Biotechnology Co., Ltd</td>
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			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JB-P5</td>
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			<td>Fiji: ImageJ</td>
			<td>National Institutes of Health, USA</td>
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			<td>Thermo scientific</td>
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			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JB-L5</td>
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			<td>Leagene</td>
			<td>DH0020</td>
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			<td>Hydrochloric Acid Solution</td>
			<td>Sichuan Xilong Science Co., Ltd</td>
			<td>210608</td>
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			<td>ImageJ2 Plugin</td>
			<td></td>
			<td>BoneJ 7.0.16</td>
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			<td>Medical Gauze</td>
			<td>Shandong Ang Yang Medical Technology Co.</td>
			<td></td>
			<td></td>
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			<td>Mersilk 3-0 Silk Braided Non-Absorbable Sutures</td>
			<td>Ethicon, Inc.</td>
			<td>SA84G</td>
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			<td>Needle Holder</td>
			<td>Jinzhong surgical instrument Co., Ltd</td>
			<td>J32010</td>
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			<td>Neutral Balsam</td>
			<td>Sinopharm Group Chemical Reagent Co., Ltd</td>
			<td>10004160</td>
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			<td>Oven</td>
			<td>Shanghai Yiheng Scientific Instruments Co., Ltd</td>
			<td>DHG-9240A</td>
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			<td>PANNORAMIC Digital Slide Scanners</td>
			<td>3DHISTECH Ltd.&#160;</td>
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			<td>PBS buffer</td>
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			<td>Povidone-iodine solution 5%</td>
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			<td>Scalpel</td>
			<td>Quanzhou Excellence Medical Co., Ltd</td>
			<td>20170022</td>
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			<td>Scan &#38; Browse Software</td>
			<td>3DHISTECH Ltd.&#160;</td>
			<td>CaseViewer2.4</td>
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			<td>Single-Use Sterile Rubber Surgical Gloves</td>
			<td>Guangdong Huitong Latex Products Group Co., Ltd</td>
			<td>22B141EO</td>
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			<td>Sodium Chloride Solution 0.9%</td>
			<td>Sichuan Kelun Pharmaceutical Co., Ltd</td>
			<td></td>
			<td></td>
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			<td>Sterile Hypodermic Syringes for Single Use</td>
			<td>Shandong Weigao Group Medical Polymer Products&#160; Co., Ltd</td>
			<td></td>
			<td></td>
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			<td>Sterile Medical Suture Needles</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., Ltd.&#160;</td>
			<td>PW8068</td>
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			<td>Tissue Processor</td>
			<td>Thermo scientific</td>
			<td>STP420 ES</td>
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			<td>Tissue Spreading and Baking Machine</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JK-6</td>
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			<td>Tribromoethanol</td>
			<td>Nanjing Aibei Biotechnology Co., Ltd</td>
			<td>M2920</td>
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			<td>Wax Trimmer</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JXL-818</td>
			<td></td>
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			<td>Xylene</td>
			<td>Chengdu Kolon Chemical Co., Ltd</td>
			<td>2022051901</td>
			<td></td>
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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.

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

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Our research focused on the effects of traditional Chinese medicine, or TCM, on osteoporosis. We are trying to explore the mechanism, efficacy, and safety of TCM intervention for osteoporosis. Micro-computed tomography imaging has recently become a popular technique for bone morphology assessment.It can provide accurate and comprehensive data on bone structure and density parameters, such as bone volume, fracture thickness, and separation. The skeletal system is the most unique tissue structure in terms of the physical properties of the body. It contains the inorganic component arginine calcium phosphate, and it is hard and stiff as a result.The process of hurting bone symptoms is tedious. We provided a complete and detailed process from modeling to osteoporosis in evaluation for researchers. We believe the application of the technology will undoubtedly bring great improvements to osteoporosis research in the context of ethnomedicine exploration.

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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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.

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