Two Orthopaedic specialists developed a list of potential keywords. A formal search strategy was then developed by an M.D. and another literature expert, and the literature was searched for concepts including osteoporosis, sarcopenia, and muscular dystrophy.

The search was completed on September 1, 2023 with no language restrictions. We also manually searched the reference lists of included articles.

①Time frame for the literature search:The time frame for the literature search was 2000-2023.

②Search databases:PubMed, Embase database, China Knowledge Network, Wanfang data platform and Vipshop journal platform.

③Search terms English search term: “osteosarcopenia; Sarcopenia-osteoporosis; Sarcopenia;osteoporosis; Animal models “; Chinese search term: “肌肉减少症;肌少-骨质疏松症; 骨质疏松症; 肌少症; 动物模型”.

Criteria for inclusion:

① The article must be about modeling or experimentation with animal models and have final modeling results; ②If it is a duplicate modeling method, choose the modeling method reported in the latest published literature.(For example, two articles from the same experimental team, with exactly the same modeling method, the most recently published article would be chosen); ③ Published in any language (studies that were not published in English were translated using Google Translate. If the article is published in Chinese, it should be at least in a scientific or technical core journal). ④ The model animals should be involved in sarcopenia and osteoporosis.

Literature exclusion criteria:

① Literature that does not match the research(For example, human studies rather than animal model studies;systematic reviews or meta-analyses); ② Duplicate content, ③Articles that cannot be downloaded in full(It’s not a subscription issue, it’s just having the title or comments and not being able to download the full text). ④ No final modeling results.

Manual searches were again conducted through the computerized searches described above. When disagreements arose, consensus was reached by two members; if consensus could not be reached, a third member was involved. The selected articles were then reviewed again for full-text relevance by two panelists.

One reviewer screened the data from the selected articles for extraction, and the results were verified by a second reviewer. Data elements extracted included title, author (year), language, modeling method and results. This scoping review focuses on modeling methods and their advantages and disadvantages.Therefore, we did not extract information about the results of the studies, nor did we assess the quality of the included studies.

We conducted a literature review based on data extracted through descriptive statistics. The benefits and drawbacks of the different modeling techniques are outlined and summarized.

Of these, 10, 392 articles were excluded at the title/abstract review stage ( Figure 1 ). Of the 4938 articles selected for full-text review, we excluded 4018. There were 2 full-text articles that we could not locate. After full-text review, we retained 38 articles for data extraction.

A search was conducted for all the identified keywords, and their number of relevant publications per year is progressively increasing. Among them, osteoporosis modeling has been researched extensively. The “OS” as a recent terminology has shown an uptick in its frequency of use over the years, albeit relatively less than other related keywords. ( Figures 2 – 5 ).

Observational indices differ between models, but typically rely on detecting muscle and bone loss. The table format highlights key aspects of these changes, providing a crucial foundation for OS models ( Table 1 ).

Aging is the most common and currently the leading clinical cause of OS. In this section, we provide an overview of several mouse models of aging linked to OS, and assess their respective merits and demerits ( Table 2 ).

First, the natural aging rat model has a lifespan of about 2.5-3 years, and old age is usually after 20 months, which is equivalent to after 55 years of age in humans. It is generally recognized that OS may be observed after 18 months of age. However, due to the high time cost of culturing naturally aging rats, further studies and reports are needed for this disease.

In studies of artificially intervened, pro-aging or premature aging models, Kondratov (9) observed specific strains of Bmal1 knockout mice (Bmal1 KO) by disrupting circadian rhythms and found that Bmal1 KO mice had a shortened lifespan and exhibited features of late aging with significant changes in skeletal structure as well as various other lesions. These mice also exhibited significant skeletal muscle weakness and structural pathological changes with altered myofilament structure and abnormal mitochondrial volume and function. One of the earliest models of accelerated senescence was the inbred strain of the accelerated senescence propensity (SAMP) mouse (10), which is now hypothesized to be due to elevated levels of oxidative stress in tissues and mitochondrial dysfunction, leading to senescence. Certain SAMP strains have been found to have musculoskeletal defects, mainly SAMP6 and SAMP3, which exhibit senile osteoporosis and degenerative temporomandibular joint disease, respectively, at approximately 3 months of age (11). Borbélyová (12) selected galactose-sensitive Wistar rats for inbreeding to create genetic models of accelerated aging and related diseases (OXYS rats), and currently their team has the 103rd generation of OXYS rats, which, in addition to cataracts, are characterized by osteoporosis, arterial hypertension, sarcopenia and neurodegenerative changes in the brain. Thus, this rat model can be used not only to study the mechanisms of aging and the pathogenesis of age-related diseases, but also to objectively evaluate new therapeutic and preventive approaches.

Human progeria and progeroid syndromes are genetic disorders and accelerated aging due to loss of DNA repair activity is one of the more studied bases of progeria models.Alyodawi K (13)found that Ercc1Δ/- progeria mouse model is similar to naturally aging rodents and has several significant features at the same time (e.g. sarcopenia, osteoporosis, degeneration of liver and kidney function, neuropathy, etc). The mechanism is due to mutations in the Ercc1 subunit of the XPF-ERCC1 endonuclease they carry. XPF-ERCC1 nucleases are involved in nucleotide excision repair, interstrand crosslink repair and double-strand break repair, and XPF-ERCC1 plays an important role in many types of DNA repair. Thus, patients with XPF mutations exhibit significant signs of accelerated aging. There is evidence that complete deletion of Ercc1 or XPF results in dramatically accelerated aging and a significantly shorter lifespan (only 1 month) in mice. However, in Ercc1-/Δ mice, aging is accelerated when XPF/Ercc1 is expressed at only 5%-10% of normal levels.

Analysis of different models of aging, including a mouse model of human progeria, suggests that treatment of naturally aging mice with certain compounds that extend healthy lifespan and/or longevity improves pathology in models of accelerated aging.Pathways such as DNA damage, oxidative stress, and stem cell dysfunction all play a role in accelerating and driving the musculoskeletal aging process. These findings clearly demonstrate the utility of therapeutic approaches to slow musculoskeletal aging. By using conditional transgenic models that selectively accelerate aging by targeting specific genetic defects, such as loss of DNA repair and reduced nuclear structure or chromosome segregation, it is possible to assess the non-cell-autonomous effects of specific tissues (e.g. bone or skeletal muscle) on the aging of other tissues.

A single mouse model has not yet emerged that fully represents all aspects of biological aging. However, detailed transcriptomic, proteomic, and metabolomic analyses suggest that among the many models of accelerated aging, the Ercc1-/Δ mouse model best reflects natural aging, including aging of the musculoskeletal system. The use of these whole-body accelerated aging models, combined with tissue-targeted aging mouse models, will not only accelerate the understanding of the mechanisms driving musculoskeletal aging, but will also facilitate the development of drugs that can prolong musculoskeletal health.

It is well known that androgen deficiency leads to osteoporosis and sarcopenia. Myokine, a humoral factor secreted by skeletal muscle, has received attention as a key factor in the interaction with muscle and bone ( Table 3 ). Orchiectomy, the surgical removal of testes, is performed on male rodents to create a model of hypogonadism-related sarcopenia and osteoporosis. This model helps researchers understand the role of androgens, such as testosterone, in the regulation of muscle and bone health and evaluate the efficacy of androgen replacement therapies. Iemura S (14) show that androgen deficiency increases the expression and secretion of Dickkopf (Dkk) 2, an inhibitor of Wnt/β-linker signaling, which is essential for maintaining muscle and bone function, in the muscles of mice. The research team initially demonstrated that Dkk2 plays a role in muscle and bone alterations in androgen-deficient mice. In vivo experiments revealed a negative correlation between serum Dkk2 levels and bone mineral density in mouse tibial trabeculae. In vitro experiments have shown that testosterone suppresses Dkk2 mRNA levels in rat muscle cells. It is therefore hypothesized that the myokine Dkk2, which connects muscle and bone, may be linked to muscle atrophy and bone loss resulting from androgen deficiency.

Borbélyová V (15) conducted a study in which they randomized male rats into control and gonadectomy groups. The male rats underwent gonadectomy on the 47th day of life and were then raised for up to 30 months. The results of the study showed that prolonged androgen deficiency resulted in reduced muscle mass compared to the control group. Androgen deficiency lasting more than two years resulted in severe loss in muscle mass and slower fat mass gain in older rats. This long-term hypogonadism model can simulate male patients with OS and offers a greater research advantage than the short-term hypogonadism experimental model currently used in young animals.

Osteoporosis in postmenopausal women is currently a widely researched issue. However, often only the reduction in bone mass following menopause is acknowledged, with little attention given to the loss of muscle tissue. Gomes RM (16) considered the ovariectomised (OVX) rat as an animal model to simulate the postmenopausal situation. The most common postmenopausal effects are associated with the malignant triad, including loss of bone and muscle mass and increased body fatness. The research team randomized female Wistar rats into non-ovariectomized rats (Sham), OVX rats, 17β-estradiol (HR)-treated OVX group, and OVX-trained group (TR). At 70 days of age, the OVX group underwent bilateral ovariectomy to successfully establish an OS model in the rats. The study found that estrogen deficiency plays a key role in bone and muscle loss. They concluded that bone density, muscle strength and muscle mass can be improved by strength training to prevent or ameliorate muscle loss and bone loss due to reduced ovarian hormone levels. A recent study in China reported that Jiangtao Ma (17) applied OVX combined with intraperitoneal injection of dexamethasone to establish a rat model of OS. After three months, BMD, grip strength, and muscle content were measured, and the OS rat model was successfully prepared. The researcher concluded that this composite modeling method offers several advantages, including the short modeling period, stable model, operational feasibility, and low cost. And the intervention was carried out by Chinese herbal bone strengthening and pain relief formula, which was considered to have a certain therapeutic effect on OS (18).

Chemical injections are often used in the laboratory to establish animal models, with the most common chemical being glucocorticoids (GC), and it has been found that overdose of GC can induce bone loss, muscle atrophy, and decreased immunity, so many researchers have used this as a starting point to employ a hormone-induced OS model ( Table 4 ). Gasparini SJ (19) conducted a study to compare the effects of two methods of corticosterone (CS) delivery in a rodent model. One was the delivery of CS (at concentrations of 25-100μg CS/mL) via drinking water and the other was the delivery of 1.5 mg CS via surgically implanted slow-release pellets for a total of 4 weeks of experimentation. It was found that both methods induced typical adverse outcomes such as fat accumulation, insulin resistance, muscle loss and osteoporosis. The team concluded that this method is safe, inexpensive, easy to regulate, and avoids stressful handling of the animals. And in a study by PAL (20), they selected male SD rats and administered 5 mg/kg of methylprednisolone (MP) daily via subcutaneous injection for 4 weeks. At the end of the treatment, they examined bone density, muscle content and micro-crt and successfully modeled osteoporosis.Mito T (21) used animal models of diabetes mellitus (DM) to track metabolic changes in skeletal muscle and bone to provide important insights into the management of diabetic complications. They used streptozotocin (STZ) to induce a type 1 DM mouse model and then applied dual-energy X-ray absorption (DXA) to determine their fat mass, skeletal muscle mass (lean body mass), bone mineral density, and bone mineral content. In the DM model, STZ administration resulted in elevated blood glucose levels, increased water and food intake, and weight loss. Anterior tibial (TA) muscle weights were measured after 30 days and tibial micro-structure was quantified by micro-computed tomography imaging in DM mice. The TA muscle weights were significantly lower in DM mice than in control mice. Furthermore, the trabecular bone volume fraction, trabecular thickness, trabecular number, and cortical thickness were substantially reduced in mice with diabetes mellitus. The method employed successfully established a mouse model of osteoporosis, presenting a novel approach to investigating diabetic complications.

Disuse muscle hypo-osteoporosis is more common in clinical settings such as prolonged bed rest (22), immobilization after motor neuron disease (23), treatment of fractures with splints or casts, or exposure to microgravity during space flight (24). In particular, such models can be better prepared for patients with OS who are bedridden for long periods of time after fracture surgery, hemiplegia, or cerebrovascular disease, when lack of stressful stimulation and weightless activities lead to bone loss and muscle atrophy ( Table 5 ). Most studies (25–31) have found that exercise improves muscle mass, strength, and function, and therefore may be protective against sarcopenia by increasing muscle mass and strength and improving mobility, while less active individuals have an increased risk of developing sarcopenia or increasing its severity.

Despite heterogeneity among studies, most studies suggest that weight-bearing and resistance exercise are most effective in the prevention and treatment of osteoporosis in older adults (32–36). The relationship between osteoporosis and sarcopenia makes sense in the context of the bone-muscle subunit. Both tissues are derived from a common type of mesenchymal projection stem cells. Muscle cells secrete bone-regulating cytokines, whereas bone cells secrete Insulin-like growth factor 1(IGF-1), which has potential muscle-stimulating properties (37).

Suspension of the hind limb by tail suspension or sling is the most commonly used method to induce a disused model (38). Tail suspension fixation is an effective model of hindlimb disuse. Unloading or immobilization models are used to study the effects of disuse on muscle and bone. This can be achieved through methods such as tail suspension, hindlimb unloading, or casting in rodents. These models assist researchers in exploring the mechanisms of disuse-induced muscle atrophy and bone loss, while also evaluating potential interventions to mitigate these effects.du F (39). Prepared an OS model by tail suspension (TS) and one hind limb (Im) fixed by plaster-casting. Their team suspended female Sprague-Dawley rats by their tails for 6 weeks. Significant changes in muscle atrophy were found at 4 weeks, and after 6 weeks the muscle fiber type of the rats was measured and a shift from oxidative to glycolytic metabolism was found in the finger extensor muscle fibers. The rationale for the suspension OS model is thought to promote muscle atrophy by disrupting multiple cellular processes, such as induction of oxidative imbalance, mitochondrial dysfunction, intercellular interactions, and abnormal protein synthesis/degradation.

Palus S (40) concluded that plaster fixation is the most commonly used model to study muscle atrophy because it simulates plaster fixation after fracture, wraps the leg with a plaster bandage or spiral wire, and allows for prolonged immobilization. Loss of bone mass is observed along with muscle atrophy, which is consistent with what is observed clinically, and its believed that this model can be used to assess muscle loss. Botulinum toxin (BTX) is now also used in the preparation of disuse models. Brent MB (41) conducted a study to establish a model of disuse OS by injecting 4 IU of BTX into the muscle tissue of the right hind limb of 12-14 week old female Wistar rats. After 6 weeks of intervention, the research team looked at the preventive or ameliorative effects of intermittent parathyroid hormone (PTH) and growth hormone (GH), alone or in combination, on wasting bone loss and muscle loss in rats. The results of the study found that PTH completely counteracted fixation-induced loss of skeletal microarchitecture, density, and distal femoral bone strength.GH increased muscle mass but did not prevent fixation-induced bone loss. However, the combination of PTH and GH increased distal femoral bone strength, skeletal microarchitecture, density, and bone density throughout the femur, as well as increasing muscle mass. Overall, the combination of PTH and GH was more effective in preventing abolition-induced bone deterioration than was its application alone. In addition, GH alone or in combination with PTH attenuated wastage-induced loss of muscle mass. Thus, the combination of PTH and GH is more effective than its application alone.

Genetic engineering, also known as gene splicing technology and DNA recombination technology, is based on the theory of molecular genetics and the modern methods of molecular biology and microbiology, to construct in vitro hybrid DNA molecules from different sources according to a predesigned blueprint and then introduce them into living cells to change the original genetic characteristics of organisms, obtain new varieties and produce new products. The purpose of genetic engineering is to modify the genetic characteristics of organisms, to obtain new varieties, and to produce new products. Depending on the technical approach, genetically engineered animal models can be classified as transgenic animal models, knockout/knock-in animal models, and genetically engineered disease animal models ( Table 6 ). Sataranatarajan K (42) suggested that aged wild-type mice usually do not show the OS phenotype until late in life (26 months or longer). For this reason, the team used genetic engineering to prepare Sod1-/- mouse models that exhibit many of the pathological features of aging at approximately 8 months of age, such as loss of muscle mass and strength, loss of skeletal muscle innervation, and increased skeletal muscle protein catabolism, which can be used to study aging-related sarcopenia and osteoporosis. The rationale is that mitochondrial dysfunction and increased hydroperoxide production induce skeletal muscle atrophy. Mito T (21) suggested that the phenotypic profiles associated with aging and mitochondrial disease appear to overlap. The research team conducted a comparative study on aging mice using a mitochondrial disease mouse model (mtDNA-deficient mitochondrial mitotic mice). Their findings revealed that osteoporosis and sarcopenia were observed in all mice upon comparing bone density and quadriceps muscle atrophy. This confirms that mtDNA mice exhibit phenotypes similar to those observed in the aging population (sarcopenia, osteoporosis).Clinical OS does not only occur in the elderly population. Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy in children. In addition to skeletal muscle, DMD also has significant effects on the skeleton. methods for the preparation of animal models of DMD are becoming increasingly sophisticated, but the pathogenesis of skeletal abnormalities in DMD remains unknown. Recently, Hey HWD (43) identified a novel bone regulatory cytokine, fibroblast growth factor-21 (FGF-21), which is significantly up regulated in skeletal muscle in animal models of DMD. The hypothesis that muscle-derived FGF-21 negatively affects bone homeostasis in DMD was proposed and experimentally investigated by using dystrophin/pro-ovalbumin double knockout (dKO) mice. The final study showed that dystrophic skeletal muscle expresses and secretes significant levels of FGF-21, which negatively regulates bone homeostasis and is an important pathological factor in the development of bone abnormalities in DMD. The study highlights the importance of muscle/skeletal crosstalk via muscle-derived factors (myokines) in the pathogenesis of bone abnormalities in DMD.

A good disease model should have the following characteristics: ① able to reproduce the human disease to be studied; ② animals can repeatedly produce the disease, which can be standardized; ③ complete background information of the animals, the life cycle to meet the needs of the experiment; ④ the animals should be inexpensive, adequate sources, easy to transport; ⑤ as far as possible, the use of small animals (44).

The most commonly used model animals in medical research are rodents, and the mouse genome is similar to that of humans and is easy to manipulate (45).

OS becomes more common with age and is associated with muscle weakness, disability, falls and fractures, and increased morbidity and mortality (46–50). Therefore, most of the old rats or prematurely aged rats are used for model preparation, and 3-6 months old rats are currently considered by researchers as the best period to observe bone microstructural changes after de-ovulation, and are a more ideal experimental animal model to study postmenopausal osteoporosis (51). However, some studies have shown that rhesus monkeys are the best model for human sarcopenia. Unlike rodents, where significant muscle mass loss occurs quickly later in life (52), the kinetics of sarcopenia in rhesus monkeys match those of humans, with onset in middle age and gradual reduction thereafter. The reduction in muscle fiber cross-sectional area significantly contributes to muscle mass loss and an age-dependent increase in muscle fibers with mitochondrial enzyme abnormalities due to mitochondrial DNA deletion mutations has been observed (45, 53). In addition, skeletal muscle accounts for a greater proportion of total body weight in primates compared to rodents and is a huge consumer of energy expenditure.

Most of the evaluation indices of animal models of OS are analyzed and evaluated from both muscle and bone aspects together, whereas animal models of sarcopenia are tested at muscle mass, muscle strength and cellular level. Muscle mass can be responded by radiography (CT, MRI), electromyography; muscle strength is most commonly tested by the test of limb grip strength, and at the molecular biology level, the general test of myogenic cell proliferation or myotubular formation speed methods.

The assessment of osteoporosis in skeletal tissue can be accomplished through multiple approaches. Measuring bone mineral density is a direct and easily observable method to verify if the index is in line with osteoporosis diagnosis. Alternatively, Micro-CT analysis can be conducted to further examine bone morphology. Lastly, the application of HE staining can facilitate an observation of the condition of bone cells and the microstructure of bone trabeculae following the material extraction.

Combining the above methods of preparing models, the author believes that the most commonly used and proven methods in preparing OS are the aging model and the chemical drug injection model.

The following is a more detailed analysis:

①Pathological Characteristics Simulation: Aging Model and Chemical Injection Model are two effective methods for simulating pathological features. The Aging Model mimics the natural ageing process in humans and accurately simulates the pathological features of sarcopenia and osteoporosis, resulting in study findings that are consistent with real disease. The Chemical Injection Model, on the other hand, uses specific drugs such as hormone inhibitors to accurately simulate the features of muscle loss and osteoporosis in model animals, leading to similar pathological manifestations.

②Controllability : Aging Models: In aging models, the pathogenesis of the model can be controlled by selecting experimental animals of varying ages. By regulating the age of the experimental animals, one can observe and analyze the changes of OS at different stages, thereby exploring the pathogenesis at varying points in time. Chemical injection model: The degree of sarcopenia and osteoporosis can be precisely controlled in model animals by regulating the dosage and frequency of drug injection. Researchers are able to select the appropriate dose and adjust the rate of disease progression by controlling the duration of drug administration.

③Reproducibility: The aging model displays natural age-related changes in the senescence model, allowing for good reproducibility when using the same animal lines and age ranges in various laboratories and research conditions. This facilitates comparison and validation of research outcomes among different laboratories. The reproducibility of chemical drug injection modeling can be improved by controlling the type of drug, dosage, and injection method. The identical pharmaceutical and injection protocol may yield comparable pathological influences in various laboratories, thereby enabling dependable assessment and authentication of research outcomes.

④Cost-effectiveness: Aging Model: Compared to some other models of OS, the aging model necessitates neither complex gene editing nor special feeding conditions, resulting in lower research costs. Chemical Injection Model: This model is relatively simple, as it only requires the selection of appropriate drugs and dosages, and its cost-effectiveness makes it suitable for research purposes.

By combining the benefits of the aging model and the chemical drug injection model, a more detailed comprehension of the pathogenesis and influencing factors of OS could be attained. Selecting the suitable method from these two models, depending on the specific experimental requirements and research goals, can enhance the dependability of the outcomes and facilitate further exploration of the disease.