Studies have demonstrated that examining the internal fixation of osteoporotic (OP) vertebrae is crucial for comprehending bone mechanics and the advancement of innovative and superior implant designs (Wang and Guo, 2020). In mechanical experiments concerning spinal fusion and instrumentation techniques, the trial production of surgical instruments necessitates extensive testing on various animal models. Consequently, the accurate selection and preparation of an exemplary animal model for OP experiments, capable of providing numerous reproducible tests, are fundamental to conducting biomechanical studies on osteoporosis. Human spinal specimens and those from non-human primates, such as orangutans, are often considered. However, their high cost, limited reproducibility, and ethical concerns significantly restrict their application as experimental models (Wang and Guo, 2020; Wilke et al., 2023).

Swartz et al. (1991) established that the calf spine possesses tissue density, equivalent mineral density, apparent density, ash density and content, compressive modulus, and strength that are analogous to those of a young, healthy human spine. Moreover, the lumbar vertebrae are sufficiently large for the assessment of spinal implants (Riley et al., 2004). As a result, in the realm of biomechanical research, the calf spine is frequently employed as an alternative to human specimens for both in vivo and in vitro experiments, including spinal fusion and instrumentation techniques (Sait et al., 2016; Akgül et al., 2020; Thomas et al., 2011; Chaudhari et al., 2011).

The preparation of calf osteoporosis (OP) models encompasses various techniques such as gene knockout (Alonso-Perez et al., 2018), hormone induction (Chen et al., 2017), low-calcium diets, and oophorectomy (Rondanelli et al., 2021). However, these methods typically require over 6 months to develop, which, when combined with the procurement of live animals, their maintenance, sterilization, and pharmacological manipulations, leads to an extended modeling cycle and a complex process. The limited reliability of these models (Chen et al., 2017; Rondanelli et al., 2021; Eastell et al., 2019) falls short of the demands for repeated animal experiments, particularly for the biomechanical assessment of spinal instrumentation within a short timeframe using a large number of animal models. In light of this, this study focused on calf vertebrae and employed the decalcifying agent ethylene diamine tetraacetic acid (EDTA) to decrease the mineral content of bovine vertebrae (Sultan and Jayash, 2023). By doing so, we aimed to alter the bone mineral density (BMD) and biomechanical properties, thereby accurately mimicking the spine of an osteoporotic patient in a biomechanical context.

Biomechanical in vitro testing plays a crucial role in the preclinical evaluation of new implants (Wang and Guo, 2020). Animal models are frequently employed as an alternative or supplement to human specimens in these tests, aiming to reduce specimen costs and variability (Wilke et al., 2023). Among various animal models, the calf lumbar spine is often selected for studying the stabilizing effects of spinal instrumentation systems due to its similarity to human spine characteristics in both quasi-static and cyclic tests (Sunni et al., 2020; Cotterill et al., 1986). Thus, in this study, the calf lumbar spine was utilized as a surrogate for the human spine in biomechanical research.

In the present study, an 18.3% mmol/L EDTA solution was employed to remove calcium and certain minerals within the vertebral body, thereby facilitating decalcification and a reduction in bone biomechanical properties. The neutral pH of the EDTA solution, coupled with its slower decalcification rate compared to acidic solutions like hydrochloric acid, offers greater stability and accuracy in binding with calcium, causing minimal tissue damage and preserving tissue cell structure integrity (Freiet al., 2022). Decalcification progress in this study showed a gradual decrease in BMD, with a slower rate initially, a more rapid change between weeks 2 and 4, and a subsequent slowdown. This may be related to the threshold of the EDTA decalcification process, that is, when a large number of minerals in the bone are chelated and free, decalcification is accelerated, and in the later stage, the change in BMD tends to be slow due to the small amount of mineral remaining. BMD of the decalcification group decreased by approximately 12.56% in the first week and 21.68% in the fourth week post-intervention. By the sixth week, BMD was 28.5% lower than pre-decalcification levels, meeting the diagnostic criteria for OP. After 8 weeks, all vertebrae in Group B reached osteoporotic levels, with an average BMD decrease of 48.9% compared to pre-decalcification values. This indicates that varying degrees of OP models can be replicated by adjusting the duration of EDTA immersion, reducing BMD by approximately 10%–50% over 1–8 weeks.

In the context of bone microstructure, the structural parameters of the trabecular bone are intimately linked to mechanical properties (Amirtham et al., 2020). Bone loss alone does not fully account for the decreased bone strength and increased brittleness observed in OP patients. Studies have revealed significant changes in trabecular structure, even with minor alterations in the trabecular volume fraction (Guenoun et al., 2017). Notably, the main trabeculae in calf lumbar spines are concentrated along the longitudinal axis, similar to humans, and are crucial for weight-bearing. Studies on vertebral trabeculae in OP patients have found that while horizontal trabeculae are shorter and thinner than vertical trabeculae, they contribute more significantly to bone structure strength (Amirtham et al., 2020). As bone mass decreases, horizontal trabeculae are preferentially lost, leading to excessive loads on the remaining vertical trabeculae and subsequent compensatory hypertrophy (Lee et al., 2011). Micro-CT analysis after 2 months of decalcification showed significantly lower values for Tb.Th, Tb.N, and BV/TV in the decalcified group compared to Group A, with higher increases in BS/BV, Tb.Sp, TBPf, and SMI. These results indicate a significant BMD reduction in Group B, with trabecular structural parameter changes possibly related to trabecular perforation and the discontinuity of the trabecular meshwork. The higher TBPf value in Group B after 2 months confirms EDTA’s role in reducing trabecular bone interconnection (Cesar et al., 2020). The loss of trabecular connectivity leads to a disproportionate loss of cancellous bone relative to cortical bone, an area of importance in OP fracture risk studies (Ozan et al., 2017). Based on the abovementioned results, we can infer that the loss of bone mass elements caused by trabecular perforation is the main mechanism of structural changes in the OP model. The decrease in bone strength caused by changes in the trabecular structure may contribute more to bone loss (Lee et al., 2011).

Studies have demonstrated that the number of trabecular bones directly affects cortical bone mechanical strength and the screw fixation ability (Wang and Guo, 2020). The statistical results in this study showed a decrease in F_max for Group B’s vertebrae with prolonged decalcification time. The correlation analysis between BMD and F_max across the two groups revealed a significant positive correlation. The axial compression test for Group B’s vertebral bodies showed a 72.83% decrease in elastic modulus, a 49.24% decrease in yield stress, and a 55.72% decrease in ultimate stress compared to Group A. As expected, Young’s modulus, yield stress, and ultimate stress of calf vertebrae were strongly positively correlated with vertebral BMD, further confirming that BMD reduction directly affects the fixation strength of pedicle screws and the compressive strength of vertebral bodies. In conclusion, while bone mass is lost, the mechanical properties of trabecular bone also change, playing a significant role in increased bone fragility and fracture risk in osteoporotic patients. Studying the microstructure and composition of trabecular bone aids in accurately evaluating its mechanical properties, which is crucial for preventing osteoporotic fractures.

Several limitations to this study should be acknowledged. First, the type, size, drying degree, and environmental medium of the samples can influence experimental outcomes. Second, there is an inevitable end-plate error between the sample and the measuring instrument due to interface friction. Third, the absence of intermediate measurements during the 8-week decalcification process means that we cannot dynamically track the changes in BMD and microarchitecture. Lastly, the sample size used in this experiment is small, and further research with a larger sample size is necessary to ensure the accuracy and effectiveness of the findings.

The process of immersing the calf lumbar spine in EDTA to replicate in vivo decalcification is an effective approach for rapidly establishing an osteoporosis model suitable for spinal biomechanics research. This method offers several benefits, such as a short modeling cycle, high repeatability, and the capacity to control the level of osteoporosis, making it an advantageous technique for these types of studies.