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 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.
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.
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 °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
2. Micro-CT scanning
3. CT data analysis
4. Decalcification of bone tissue
5. HE staining
6. HE image analysis
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 differences between mice of OVX and Sham group for a number of parameters that were estimated from Micro-CT. Namely, the ratio of bone volume to total tissue volume (BV/TV) in the OVX group was 3% lower than that in the Sham group. The trabecular thickness in mice of OVX was lower than in those of the Sham group, with a relative difference percentage of 39.3%. The trabecular separation in OVX mice was greater than that in Sham mice. Figure 4 shows 3D displays of Trabecular ROIs extracted from reconstructed bone volume for each group. Compared with the Sham group (Figure 4A), the bone density of mice after ovariectomy, the trabeculae were sparse and showed osteoporosis (Figure 4B).
HE staining analysis
In addition, histopathological analysis confirmed the changes found in the Micro-CT analysis. After 8 weeks, HE staining showed that the trabecular bone (red) under the growth plate of the distal femur of mice after OVX was reduced compared with the Sham group, and there was almost no obvious thick trabecular structure, and a large number of fat-like granules appeared (Figure 5A). Based on the quantitative analysis of tissue sections, the OVX mice had less trabecular bone area than the Sham one (Figure 5B).
Figure 1: Screenshot of the interface of subvolume reconstruction. A subvolume reconstruction in the green rectangle to get down to 10 µm in a 5.12 mm x 5.12 mm field of view (FOV). Please click here to view a larger version of this figure.
Figure 2: Image mask construction in black and white. (A1–A3): HE Images from the Sham group (Scale bar = 200 µm). Bone trabeculae are shown in black within the selected region and other tissues in white. (B1–B3) Images of OVX group (Scale bar = 200 µm). Please click here to view a larger version of this figure.
Figure 3: Comparison of microstructural parameters of distal femoral trabeculae between OVX and Sham groups. (A) The ratio of bone volume to total tissue volume (BV/TV [%]) in the OVX group was lower than that in the Sham group. (B)The trabecular thickness (TB.Th [µm]) in mice of OVX was lower than in the Sham group. (C) The trabecular separation (TB.sp [µm]) in OVX mice was greater than in Sham mice. *P < 0.05. Please click here to view a larger version of this figure.
Figure 4: Representative Micro-CT images of the trabecular bone in the distal femur. Compared with the (A) Sham group, the bone density of mice after ovariectomy (B) displayed sparse trabeculae and showed osteoporosis. Please click here to view a larger version of this figure.
Figure 5: Histological analysis of the trabecular bone area in the distal femur of OVX and Sham groups. (A) Representative HE-stained images of the distal femur in each group (Scale bar = 500 µm). Black arrows show trabeculae. (B) The quantitative analysis of the trabecular bone area of the total tissue volume in the selected ROI. *P < 0.05. Please click here to view a larger version of this figure.
Parameter | Sham-operated group (SHAM) (n = 5). |
Ovariectomy group (OVX) (n = 5). |
P Value |
Bone volume–to–total tissue volume ratio (%) | 7.3 ± 0.9 | 4.2 ± 0.5 | 0.012* |
Trabecular thickness (μm) | 79.5 ± 5.5 | 53.4 ± 6.0 | 0.013* |
Trabecular separation (μm) | 212.5 ± 8.7 | 249.4 ± 8.3 | 0.015* |
Values are the mean ± SD. * Significantly different (P<0 .05). |
Table 1: Trabecular bone parameters estimated from Micro-CT.
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 been conducted using mice22. To establish an osteoporosis model in adult female C57/BL6J mice, ovariectomy is performed at 12 weeks of age, which is the optimal time for this procedure. Mice are mature and fertile from 8-12 weeks of age, and ovariectomy has a significant effect on their bone mass at this time10. Previous studies show that before 12 weeks, the bones of mice grow rapidly, and the indicators of bone morphology, BMD, and bone biomechanics increase quickly. The total BMD and cortical bone BMD are significantly correlated with the age of the mice. However, after 12 weeks, the bone metabolism of mice enters a stable period, and the above indicators tend to be stable23,24.
Bone microarchitecture has been proposed as the primary factor influencing bone fragility, independent of BMD. The bone fragility of osteoporosis is not fully explained by a deficit in bone mass. BMD can only explain 60%-70% of bone strength25. In terms of structural composition, cortical bone comprises 80% of the bone mass, whereas cancellous bone exhibits only 10% of the measurements after losing 50% of the bone mass, suggesting that bone mass measurements alone are not sufficient to assess bone mass loss. In addition to bone volume, structural changes in bone trabeculae are critical to bone strength. Cancellous bone contains hematopoietic bone marrow tissue or adipose tissue. Its surface is quite large, about eight times larger than that of cortical bone. This large surface area connected to the bone marrow allows the cancellous bone to have a fairly high bone conversion rate. That is why we chose the microstructure of bone trabeculae as the main indicator for observing osteoporotic changes. The trabecular bone tissue in the skeleton is constantly undergoing remodeling during growth, during which newly formed bone trabeculae replace the existing ones and form secondary spongy bone. Therefore, in normal conditions, morphometric analysis of trabecular bone is mainly focused on the secondary spongy bone area. Primary spongy bone tissue, on the other hand, is a congenitally present and relatively stable structure that is not remodeled and thus needs to be excluded from the analysis. Generally, the bone within a certain distance from the growth plate can be considered as actively remodeling tissue. Therefore, we selected a region of interest (ROI) that starts from 540 µm above the growth plate and spans 1600 µm in the proximal direction for the analysis. According to our findings, mice exhibit very different microstructural characteristics in trabecular regions after ovariectomy.
The Micro-CT technology used in this study is a non-destructive 3D imaging technique developed in recent decades and is gradually being applied to the pharmacological study of ethnomedicinal herbs. It provides a clear understanding of the internal microstructure of the sample without destroying it. As for the histopathological examination, the resin embedding method damages enzyme activity and protein antigenicity and cannot be reliably used for histochemistry or immunohistochemistry26. The traditional paraffin embedding can be combined with techniques such as immunohistochemistry (IHC), fluorescence in situ Hybridization (FISH), and confocal laser scanning microscope (CLSM) to quantitatively detect low-abundance substances in bone tissue at the molecular level, thereby gaining a deeper understanding of the mechanisms and regulation of bone metabolism. The panoramic slide scanner can rapidly convert slides into high-resolution digital images, making it possible to quantitatively analyze bone histomorphology data using computer software. In summary, the combination of Micro-CT and bone histomorphology can provide a more detailed and accurate evaluation of bone microstructure.
Traditionally, assessment of bone quality was largely limited to dual-energy X-ray absorptiometry (DXA), limited resolution computed tomography, or technically challenging magnetic resonance imaging27. Although the combination of the two techniques has its unique advantages, it also has its limitations. First, although Micro-CT allows for long-term continuous monitoring of the same animal, in vivo imaging of skeletal microstructures in live small animals, especially mice, is still technically challenging28. Second, the absence of a structured and internationally recognized protocol for acquiring and analyzing data from different instruments or users makes it difficult to compare datasets and reproduce research results across studies. Third, due to the characteristics of Micro-CT, exposure to ionizing radiation is inevitable. Fourth, although our simple and economical protocol can provide quantitative analysis of HE-stained bone images, it is mainly based on a semi-automatic segmentation strategy, which is more laborious than complex automated methods.
In conclusion, the application of this technology will undoubtedly bring a great impetus to osteoporosis research in ethnomedicine during the exploration process.
The authors have nothing to disclose.
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.
4% Paraformaldehyde | Biosharp | BL539A | |
Adobe Photoshop | Adobe Inc. | ||
Ammonia Solution | Chengdu Kolon Chemical Co., Ltd | 2021070101 | |
Analyze 12.0 | AnalyzeDirect, Inc | ||
Anatomical Forceps | Jinzhong surgical instrument Co., Ltd | J3C030 | |
Anhydrous Ethanol | Chengdu Kolon Chemical Co., Ltd | 2022070501 | |
Automatic Dyeing Machine | Thermo scientific | Varistain™ Gemini ES | |
Bone Microarchitecture Analysis Add-on | AnalyzeDirect, Inc | ||
C57BL/6J mice | SPF (Beijing) Biotechnology Co., Ltd. | ||
Carrier Slides | Nantong Mei Wei De Experimental Equipment Co., Ltd | 220518001 | |
Coverslips | Nantong Mei Wei De Experimental Equipment Co. | 220518001 | |
Decalcification Solution | Wuhan Xavier Biotechnology Co., Ltd | CR2203047 | |
Delicate Scissors | Jinzhong surgical instrument Co., Ltd | ZJA010 | |
Embedding box marking machine | Thermo scientific | PrintMate AS | |
Embedding Machine | Wuhan Junjie Electronics Co., Ltd | JB-P5 | |
Fiji: ImageJ | National Institutes of Health, USA | ||
Film Sealer | Thermo scientific | Autostainer 360 | |
Freezing Table | Wuhan Junjie Electronics Co., Ltd | JB-L5 | |
H&E Staining Kit | Leagene | DH0020 | |
Hydrochloric Acid Solution | Sichuan Xilong Science Co., Ltd | 210608 | |
ImageJ2 Plugin | BoneJ 7.0.16 | ||
Medical Gauze | Shandong Ang Yang Medical Technology Co. | ||
Mersilk 3-0 Silk Braided Non-Absorbable Sutures | Ethicon, Inc. | SA84G | |
Needle Holder | Jinzhong surgical instrument Co., Ltd | J32010 | |
Neutral Balsam | Sinopharm Group Chemical Reagent Co., Ltd | 10004160 | |
Oven | Shanghai Yiheng Scientific Instruments Co., Ltd | DHG-9240A | |
PANNORAMIC Digital Slide Scanners | 3DHISTECH Ltd. | PANNORAMIC DESK/MIDI/250/1000 | |
PBS buffer | Biosharp | G4202 | |
Povidone-iodine solution 5% | Chengdu Yongan Pharmaceutical Co., Ltd | ||
Quantum GX2 microCT Imaging System | PerkinElmer, Inc. | ||
Rotary Microtome | Thermo scientific | HM325 | |
Scalpel | Quanzhou Excellence Medical Co., Ltd | 20170022 | |
Scan & Browse Software | 3DHISTECH Ltd. | CaseViewer2.4 | |
Single-Use Sterile Rubber Surgical Gloves | Guangdong Huitong Latex Products Group Co., Ltd | 22B141EO | |
Sodium Chloride Solution 0.9% | Sichuan Kelun Pharmaceutical Co., Ltd | ||
Sterile Hypodermic Syringes for Single Use | Shandong Weigao Group Medical Polymer Products Co., Ltd | ||
Sterile Medical Suture Needles | Shanghai Pudong Jinhuan Medical Products Co., Ltd. | PW8068 | |
Tissue Processor | Thermo scientific | STP420 ES | |
Tissue Spreading and Baking Machine | Wuhan Junjie Electronics Co., Ltd | JK-6 | |
Tribromoethanol | Nanjing Aibei Biotechnology Co., Ltd | M2920 | |
Wax Trimmer | Wuhan Junjie Electronics Co., Ltd | JXL-818 | |
Xylene | Chengdu Kolon Chemical Co., Ltd | 2022051901 |