Impact of fluoride and aluminum co-exposure on bone growth and quality in juvenile rats: dose and duration effects.

4. Discussion

The results of this experiment revealed that as the exposure time and dosage of F and Al increased, the function of growth plate chondrocytes, metaphyseal cancellous bone osteoblasts, and osteoclasts exhibited a two-way effect, transitioning from a promotional to an inhibitory effect. Consequently, it led to a shift in longitudinal growth, bone microstructure, and bone quality from enhancement to deterioration.

The growth plate (epiphyseal plate) is crucial for the growth of long bones. Chondrocytes within the growth plate continuously undergo proliferation, differentiation, maturation, hypertrophy, and calcification to form primary trabecular bone. This bone is subsequently replaced by true bone tissue, becoming secondary trabecular bone(Pazzaglia et al., 2016). Therefore, any factor that affects chondrocytes can interfere with the longitudinal growth of bones. Current research has confirmed that F directly affects target cells. Its strong oxidative properties in vivo increase the production of free radicals, promote lipid peroxidation, and directly damage the DNA structure of cells(Johnston, 2020; Babu, 2022). In addition, F can inhibit the proliferation and differentiation of chondrocytes, promote their apoptosis through pathways such as PI3K/AKT/mTOR and Indian hedgehog (Ihh), ultimately affecting the process of endochondral ossification(Ma, 2021; Gao, 2020). Al also has a direct inhibitory effect on target cells. Al ions(Al3+) competitively bind with phosphate ions and citrate, inhibiting phosphorylation reactions. This can lead to changes in the activity of most enzymes and proteins, causing cell damage (de Sautu, 2018). Experimental evidence has shown that Al reduces the activity of chondrocytes and the synthesis of matrix collagen protein, thereby inducing cartilage formation defect (Zhang, 2017). The results of this study indicate that the effect of F and Al co-exposure on chondrocytes shifts from promotion to inhibition. This bidirectional effect is also observed in experiments where F acts alone (Qu, 2008). Our research further suggests that this bidirectional effect of F and Al on chondrocytes is not only dependent on the dose but also on the duration of exposure. It appears that the initial promoting effect of low doses or short-term exposure to F and Al is likely a result of the body’s compensatory mechanisms. However, as the dose of F increase or the exposure period extends, these compensatory mechanisms become overwhelmed. Once this occurs, the direct negative effects of F and Al on chondrocytes eventually appear.

In addition to their direct effects on chondrocytes, F and Al also influence cartilage mineralization. Elevated F ion concentrations can gradually disrupt calcium homeostasis within the body, resulting in a deficiency of calcium in the extracellular fluid. This deficiency hinders the timely mineralization of the cartilage matrix (Yu, 2021). Furthermore, Al competitively inhibits the binding of proteoglycans to matrix collagen, which lead to the formation of immature cartilage matrix. Consequently, this causes delayed calcification of hypertrophic chondrocytes in the growth plate and slows down longitudinal bone growth (Marques, 2022). In summary, our study demonstrates that increasing doses of F and Al or extended periods of exposure result in a transition from active to inhibited proliferation of chondrocytes. This shift ultimately leads to impaired bone formation in the growth plate cartilage and delays in longitudinal bone growth. The observed changes in body weight and tibia length further corroborate these findings.

After mineralization of the growth plate cartilage, blood vessels infiltrate to form primary trabecular bone (also known as cancellous bone), which is then remodeled to secondary trabecular bone through the coordinated actions of osteoblasts and osteoclasts (Touaitahuata, 2014). This remodeling process hinges on a balance between bone formation and bone resorption, regulated by numerous factors. The results of this study indicate that simultaneous intake of F and Al has a dose-dependent effect on the quantity and function of osteoblasts and osteoclasts, with low doses promoting and high doses inhibiting. In vitro studies have confirmed the bidirectional effects of F on osteoblasts, whereas Al consistently shows negative effects on osteoblasts (Zhu, 2016). The impact of F or Al on osteoclasts is more contentious. Some studies suggest that F or Al can have bidirectional effects on bone resorption, while others indicate a more pronounced increase in bone resorption with exposure to these elements (Song, 2020). Based on the information provided, we infer that when high levels of F and low levels of Al are ingested simultaneously, the effects on bone health are primarily dominated by F. High levels of F have a stimulating effect on bone formation, which can overshadow the direct inhibitory effect that low levels of Al might have on osteoblasts. Furthermore, osteoblasts may be activated by F and Al initially, leading to an increase in bone formation, which is then followed by an increase in bone resorption.

Studies have shown that the activation of osteoblasts is an early and dominant process in the development of fluorosis. Once activated, osteoblasts can stimulate osteoclasts through the PTH pathway and the OPG/RANK/RANKL network system (Koroglu, 2011). This activation may also result from direct cell signaling between osteoblasts and osteoclasts (Kim, 2020). Additionally, research has also found that F inhibits the expression of OPG in osteocytes, increases the RANKL/OPG ratio, and enhances osteoclast differentiation driven by osteocytes through the RANK-JNK-NFATc1 signaling pathway (Jiang, 2020, Jiang, 2020). Therefore, increased bone formation may stimulate bone resorption through the aforementioned pathways, distinct from the classical bone remodeling process initiated by osteoclasts. Additionally, the selection of dosage can help explain the inconsistent results often observed in in vivo and in vitro studies on the effects of F and Al on osteoclasts and bone resorption. Bone formation and resorption play important roles in the remodeling and shaping secondary trabeculae, forming the cancellous bone at the epiphyseal end of the bone. In this region, BMD primarily reflects the quantity of trabeculae and the extent of calcium salt deposition. The bone’s microstructure can be visualized as a framework composed of orderly arranged collagen fibers within the organic matrix, along with the deposition of hydroxyapatite crystals that form the inorganic framework structure. This microstructure not only reflects BMD but also reveals parameters such as the number, thickness, connectivity, and the plate-like or column-like structure of trabeculae, offering a more comprehensive reflection of changes in bone biomechanics (Mustafy, 2019). Clinical data shows that simultaneous exposure to high levels of F and Al in children can lead to bone sclerosis characterized by thick and dense trabeculae, increased bone mass, and enhanced bone mineral deposition. Signs of osteoporosis may emerge as children age (Li, 2014). This experiment’s results align with these findings, demonstrating an increase in new bone formation, leading to thicker trabeculae, greater connectivity, and higher bone hardness. The increase in bone hardness is currently believed to be due to the competition between poorly soluble fluorapatite and aluminum phosphate with calcium salts for bone deposition sites. The crystal volume of these substances is larger than that of normal calcium phosphate, leading to an increase in the average width of hydroxyapatite crystals (Riedel, 2017). However, there is a negative correlation between crystal width and the stress resistance of long bone fractures. Accelerated bone turnover, driven by active bone resorption, may replace normal hydroxyapatite structures, ultimately reducing overall mechanical properties. Studies have reported that while F increases vertebral bone density, the bone strength does not increase proportionally. Even when trabecular volume remains constant, bone strength may already be diminished (Vestergaard, 2008). There are reports suggesting that Al can thicken alveolar bone in rats, and increase the proportion of organic matrix (Souza-Monteiro, 2021). Other studies indicate that although Al may help maintain bone mass, it can reduce mineralization and hardness of bone tissue, resulting in decreased bone strength. When F and Al act together, they not only disrupt the normal deposition of calcium salts but also affect the formation of bone’s organic matrix and collagen fibers. Consequently, although initial exposure to F and Al may increase bone density in growing individuals, the ongoing remodeling of the bone’s microstructure can gradually alter the normal material and structure of trabecular bone. This alteration can ultimately lead to a decrease in bone mechanical properties.

If this study can provide biochemical evidence of F and Al content in bones, and include experimental designs comparing the effects of F or Al alone, it would facilitate further analysis of the interactive results and mechanisms of F and Al. It is hoped that this can be supplemented and improved in subsequent experiments. However, in fluorosis-endemic areas, where minors are increasingly exposure to dietary Al, the findings of this research still hold significant reference value. They serve as a reminder of the potential skeletal damage caused by long-term concurrent intake of F and Al.

5. Conclusions

In conclusion, F and Al exert a dual effect on the longitudinal bone growth and bone quality of growing rats by directly or indirectly affecting the functions of osteoblasts, osteoclasts, and chondrocytes. While excessive environmental exposure to F and Al may initially promote bone formation and increase bone density, this effect comes at the expense of normal bone microstructure and material. Consequently, this can lead to a decline in the biomechanical properties of bones over the long term.