Abstract
Background/Aim: To investigate the feasibility of establishing a mandibular osteosarcoma model in Sprague-Dawley (SD) rats using tissue block transplantation, providing a foundational model for osteosarcoma research. Materials and Methods: Fourteen male SD rats, 3 weeks old and SPF grade, were randomly divided into a control group (n=4) and a mandibular osteosarcoma group (n=10). Using tissue block transplantation, UMR106 cell-induced tumor tissues were transplanted subcutaneously into the left mandibular marrow cavity of the SD rats. Observations included behavioral changes, weight variations, tumor growth, and tumor formation rate. Bone changes were monitored via micro-CT scanning, and histological analysis was conducted using HE staining. Results: Two weeks post-transplantation, the mandibular osteosarcoma group exhibited significant left facial swelling, malocclusion, eating difficulties, and weight loss compared to the control group. The tumor formation rate was 80% (8/10). Micro-CT scans indicated significant bone destruction in the osteosarcoma group. HE staining revealed high cellular atypia and pathological mitoses in both subcutaneous and mandibular osteosarcoma cells, with no notable abnormalities in lung tissues. Conclusion: Tissue block transplantation is a viable method to establish a mandibular osteosarcoma model in SD rats. This method is simple, with a high tumor formation rate, providing an ideal animal model for mandibular osteosarcoma research.
Osteosarcoma (OS) originates from primitive osteogenic mesenchymal cells and consists of malignant osteoblasts producing immature bone or osteoid tissue. It is the most common primary malignant bone tumor in adolescents (1, 2). Based on the site of occurrence, OS can be classified into jaw osteosarcoma (JOS) and long-bone osteosarcoma (LBOS). JOS is relatively rare, accounting for only 5%-10% of all OS cases, primarily affecting the mandible (3, 4). There are several clinical and biological differences between JOS and LBOS (5). Although JOS has a lower rate of lung metastasis and a better prognosis compared to LBOS (6, 7), the prognosis of patients with advanced and recurrent OS has not significantly improved over the past 30 years (8, 9). Therefore, suitable animal models are needed to replicate the complexity and heterogeneity of OS. Animal models of OS often use immunodeficient experimental animals, which may not accurately simulate the tumor microenvironment (10-12). Research has largely focused on mouse LBOS models, with only one report on a JOS model, highlighting the urgent need for research on JOS animal models (13, 14). Given the significant differences between the innate and adaptive immune systems of mice and humans (15, 16), SD rats, with their larger size, ease of surgical manipulation, reproducibility, and intact immune system, are considered an ideal model for OS. To our knowledge, there are currently no reports on the SD rat JOS model. Therefore, this study aimed to investigate the feasibility of establishing an SD rat JOS animal model using tissue block transplantation, to provide a foundational model for exploring new treatments and studying the immune microenvironment of JOS.
Materials and Methods
Experimental animals and cell lines. The Department of Experimental Animals at Kunming Medical University provided five 4-week-old SPF-grade male nude mice weighing (15±5) g and fifteen 3-week-old SPF-grade male SD rats weighing (50±5) g. The rats were randomly divided into a control group (n=4) and a mandibular osteosarcoma group (n=10). The osteosarcoma cell line UMR106, sourced from SD rats, was provided by the Key Laboratory of Stomatology at Kunming Medical University.
Establishing the osteosarcoma model using tissue block transplantation. Subcutaneous osteosarcoma in nude mice. Logarithmic phase UMR106 cells (106/0.2 ml) were inoculated subcutaneously into the right dorsal area of the nude mice. Tumor growth was monitored every two days post-operation. Once the tumors reached approximately 1.0 cm × 1.0 cm × 1.0 cm, the tumor tissues were aseptically excised and cut into 1 mm3 pieces, and then stored in serum-free high-glucose medium at 4°C.
Mandibular osteosarcoma in SD rats. SD rats were anesthetized with an intraperitoneal injection of 3 mg/ml pentobarbital. After preparing and disinfecting the surgical area, a 1.0 cm incision was made from the anterior edge of the left ear base to the corner of the mouth. The skin and subcutaneous tissues were carefully dissected to fully expose the masseter muscle, facial nerve, and blood vessels. An incision was made between the buccal branches of the facial nerve, dissecting to the mandibular bone surface and separating forward to the anterior edge of the mandibular ramus. A 2 mm diameter bone defect was created using a dental drill, into which the tumor tissue block was placed. The incision was closed with bone wax and layered sutures, with erythromycin applied to the wound to prevent infection (Figure 1). The rats were kept on a warming blanket during and after surgery to maintain a body temperature of approximately 36.5°C. The control group was normally housed without any treatment. This experiment was approved by the Animal Ethics Committee of Kunming Medical University (No. kmmu20231535).
The process of establishing a subcutaneous osteosarcoma model in nude mice and a mandibular osteosarcoma model in Sprague-Dawley (SD) rats.
General observations. Daily observations were made at 9 AM, recording the rats’ mental state, eating behavior, signs of infection, and tumor growth. Weekly photographs were taken, and weights were recorded. A weight change curve was plotted at the end of the experiment.
Micro-CT scanning. The collected mandibular specimens from SD rats were subjected to high-resolution micro-CT scanning to observe changes in bone density (CT model: NMC-100).
Hematoxylin and eosin (HE) staining. Tumor samples from the subcutaneous osteosarcoma in nude mice, mandibular osteosarcoma in SD rats, and lung tissues were fixed, dehydrated, embedded in paraffin, sectioned, deparaffinized, stained with hematoxylin and eosin, treated with xylene for transparency, and sealed with neutral balsam for microscopic examination and imaging.
Statistical analysis. Statistical analyses were performed using SPSS 22.0 (IBM, Armonk, NY, USA). The main statistical tests included independent sample t-tests and one-way ANOVA. Results were expressed as mean±standard deviation (x̄ ±s). A p-value <0.05 was considered statistically significant.
Results
Osteosarcoma growth and tumor formation rate. One week after tumor implantation in nude mice, spherical, soft-textured tumors were visible (Figure 2A). By the second week, the tumors measured approximately 1.0 cm × 1.0 cm × 1.0 cm, with a 100% tumor formation rate and no spontaneous regression observed (Figure 2B). One day after the tissue block transplantation, the SD rats in the mandibular osteosarcoma group appeared in good health, with normal eating and drinking habits, stable sutures at the surgical site, and normal skin color. One week post-operation, local swelling was observed on the surgical side of the cheek, with a hard texture, no mobility, and no signs of infection (Figure 2C). Two weeks post-operation, compared to the control group, the mandibular osteosarcoma group exhibited significant facial swelling (Figure 2D), malocclusion, eating difficulties, and significant weight loss (Figure 3). All ten SD rats used for modeling survived, with two showing no tumor growth, resulting in a tumor formation rate of 80% (8/10).
Tumor growth in subcutaneous osteosarcoma of nude mice and mandibular osteosarcoma of Sprague-Dawley (SD) rats.
Weight change curve of Sprague-Dawley (SD) rats.
Gross observation. Grossly, the subcutaneous osteosarcomas in nude mice and the mandibular osteosarcomas in SD rats had distinct capsules, a hard texture, nodular surfaces, and extensive vascular distribution. Compared to the control group, no significant abnormalities were observed in the lung tissues of the mandibular osteosarcoma group.
Micro-CT scanning. micro-CT scans followed by 3D image reconstruction showed that the control group’s mandibles had intact cortical bones and symmetrical structures (Figure 4A and B). In contrast, the mandibular osteosarcoma group displayed asymmetrical mandibles due to tumors, severe bone destruction, and unclear boundaries (Figure 4C and D).
Micro-CT scanning and 3D reconstruction. Control group in Sprague-Dawley (SD) rats (A, B); Mandibular osteosarcoma in SD rats (C, D).
HE staining. HE staining revealed diffuse growth of tumor cells in both the subcutaneous osteosarcomas of nude mice and the mandibular osteosarcomas of SD rats. Tumor cells varied in size, appearing spindle-shaped or polygonal, with single or multiple nuclei, enlarged nucleoli, reduced intercellular matrix, and a high nucleus-to-cytoplasm ratio. Pinkish new bone-like tissue, granular calcification, and necrotic tumor cells were observed, occasionally with giant tumor cells (Figure 5A and B). The lung tissues of both groups exhibited a uniform reticular structure with clear edges, thin alveolar walls, and regular distribution. No thickening, congestion, or edema was observed in the alveoli (Figure 6A and B).
Hematoxylin and Eosin staining of subcutaneous osteosarcoma in nude mice and mandibular osteosarcoma in Sprague-Dawley (SD) rats. Subcutaneous osteosarcoma in nude mice (A); Mandibular osteosarcoma in SD rats (B).
Hematoxylin and Eosin staining of lung tissues in the control group and mandibular osteosarcoma group. Control group in Sprague-Dawley (SD) rats (A); Mandibular osteosarcoma in SD rats (B).
Discussion
Animal models of osteosarcoma can be categorized into spontaneous tumor models, induced tumor models, genetically engineered tumor models, and transplantable tumor models (17). Transplantable tumor models, characterized by stable biological properties, consistent growth, short experimental cycles, and high reproducibility, are commonly used in osteosarcoma research. Common experimental animals used for transplantable osteosarcoma models include nude mice, mice, rats, and zebrafish, each with distinct advantages and disadvantages. For instance, nude mice are frequently used in osteosarcoma transplantation studies due to their lack of an immune system, which allows tumors to grow without immune interference (18). Mice are cost-effective, have short experimental cycles, and exhibit consistent growth, making them a popular choice for constructing osteosarcoma models (19, 20). However, their immune systems differ significantly from humans (16). Zebrafish xenograft models have short construction cycles and are often used to study new osteosarcoma treatments and mechanisms (21, 22), but their skeletal structure and function differ from mammals (23), and they have high experimental condition requirements, limiting their application. SD rats and Wistar rats, being larger than mice, offer easier surgical manipulation, lower experimental condition requirements than zebrafish, and possess intact immune systems, making them valuable for osteosarcoma model research (24, 25).
Currently, there are no reported SD rat models of mandibular osteosarcoma. This study is the first to construct an SD rat model of mandibular osteosarcoma using tissue block transplantation, achieving an 80% tumor formation rate (8/10) without lung metastasis. Micro-CT and histological analyses confirmed that this model successfully replicated the morphological and histological characteristics of osteosarcoma. Bertin et al. (14) also reported no lung metastasis in their mouse JOS model using MOS-J cells and patient-derived tumor tissues, consistent with our findings. The two transplantation failures in our study may be attributed to necrosis, insufficient tissue quantity, or improper preservation during transplantation.
Factors affecting model success include: 1) Animal age: Three-week-old rats, with immature immune functions, had a higher transplantation success rate. Four-week-old rats, with stronger immune functions, had a lower tumor formation rate. 2) Transplantation site: The richly vascularized mandible is conducive to tumor formation. 3) Tumor block preservation: Tumor blocks should be stored in serum-free medium or PBS solution at 4°C to maintain cell viability and proliferative properties. 4) Tumor block size: Excessive inoculation leads to rapid tumor growth, while insufficient inoculation hinders tumor formation. 5) Transplantation method: Tissue block transplantation is more stable than cell suspension injection, offering higher tumor formation and lung metastasis rates (26). Recent studies indicate that cell sheet transplantation has a higher tumor formation rate than cell suspension injection (100% vs. 67%) and faster tumor growth (27, 28). Further research is needed to determine whether cell sheet or tissue block transplantation is superior for constructing rat JOS models.
Emerging therapies, such as immunotherapy, gene therapy, phototherapy, and traditional Chinese medicine offer new hope for JOS patients, but their efficacy in JOS requires further exploration and validation (29). Due to the rarity of JOS, large-scale clinical trials are challenging, highlighting the importance of establishing appropriate JOS animal models. This study successfully established an immunocompetent SD rat JOS model using tissue block transplantation, laying a foundation for subsequent research on JOS treatments and the role of the immune microenvironment in JOS. However, this study has limitations, including the exclusive focus on the UMR106 osteosarcoma cell line’s tumor formation in nude mice and SD rats. Further exploration of other osteosarcoma cell lines and patient-derived tumor tissues is needed.
Acknowledgements
This study was financially supported by the Medicine Leading Talent of Yunnan Province Health Care Committee (No.L-2018010), Yunnan Province Xing Dian Talent (No.XDYC-MY-2022-0052) and Scientific Research Fund Project of Yunnan Provincial Department of Education (No.2024Y241).
Footnotes
Authors’ Contributions
Lanlan Zhang completed the research and drafted the initial manuscript. Jiaoyan Liu and Hongrong Zhang conducted most of the animal experiments. Yemei Qian and Liqin Zhang participated in the research, while Weihong Wang supervised the project. Lanlan Zhang, Jiaoyan Liu, and Hongrong Zhang contributed equally as first Authors. All Authors significantly revised the manuscript and approved the submitted and revised versions.
Conflicts of Interest
All Authors declare that there are no conflicts of interest in relation to this study.
- Received July 15, 2024.
- Revision received August 7, 2024.
- Accepted August 16, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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