One of the main findings of this study was that age was negatively correlated with both lumbar spine and total body BMD in the study population. This finding was corroborated by the significantly smaller mean BMD z-score values, both in the lumbar spine and in the total body, in the participants aged 9.3 to 10.7 years (G2) than in the others indicating that this range is a determinant age range for bone loss. Additionally, the older DMD patients had lower (z-score ≤ -2.0) lumbar spine and total body BMD z-score values than others (> 11.2 years and > 18 years, respectively).
The correlation between age and the lumbar spine BMD z-scores was negative and moderate, while the correlation between age and the total body BMD z-score was negative and strong. Nutritional complications get worse with age . This interrelationship may be related to the mechanical load of skeletal muscles on the bones of the entire body has decreased due to the loss of muscle function over time. Larson et al.  compared the lumbar spine and the proximal femur BMD in DMD patients, and there was a significantly larger reduction in the femur BMD than in the lumbar spine BMD. Spine BMD was affected when the patients lost the ability to walk, while that of the proximal femur was low even before they lost the ability to walk.
In this study, the BMI results corroborated the susceptibility of DMD patients to overweight and obesity (Table 1). Adipose tissue is associated with the production of inflammatory cytokines, which can negatively affect bone metabolism. On the other hand, adipose tissue also has an indirect positive effect on bone metabolism by producing hormones, cytokines, and adipokines, such as circulating leptin and insulin. These substances stimulate bone formation . Given that this tissue affects bone metabolism in different ways, it is suggested that the prevalence of overweight and obesity is not the main determining factor for bone loss in the population studied.
Regarding height, larger percentages of participants with short stature were observed in the older groups, reaching 66.7% in G4. The etiology of short stature in populations with DMD is still unclear. However, previous studies suggest that low levels of growth hormones and the loss of muscle tone are partly responsible for short statures in DMD patients . Moreover, height outcomes can be predicted by genetics. Distal deletions and central mutations of the DMD gene are associated with short stature .
The weaker correlation between age and the BMD z-score of the lumbar spine compared with that of the total body may be explained by the skeleton attempting to adapt to the gravitational load associated with the excess weight of participants . DMD patients are susceptible to overweight and obesity due to the prolonged use of glucocorticoids, decreased mobility, and reduced energy expenditure, limiting their ability to perform physical activity .
The results of our study indicated that older DMD patients might have a more marked impairment in BMD than do younger patients, both in the lumbar spine and in the total body. Other studies have shown results that are like ours. In the study by Mayo et al. , there was low BMD in patients over 11 years of age. In the groups with average ages of 11 years and 14 years, the BMD z-scores of the lumbar spine were -2.4 and -3.6, which differed considerably. Regarding BMD of the total body, King et al.  concluded that DMD patients aged between 11 and 17 years had low BMD, with z-scores ranging between -3.0 and - 4.0, which were lower than those in our study.
In addition, in our study, most of the participants in G4 (83.3%) had low BMD, which may be associated with disease progression. The damage to muscle fibers present in DMD patients is related to chronic inflammation of the muscles and the release of inflammatory cytokines, which impair the function of the cells involved in the formation of bone tissue. This damage can be a contributing factor to bone loss in DMD patients .
In addition, other factors are also associated with low BMD in DMD patients. The use of glucocorticoids has already been shown to produce oxidative stress in multiple tissues, including bone. There is an association between continued use of glucocorticoids and decreased bone formation and increased bone resorption, resulting in the increased renal excretion of calcium and reduced gastrointestinal absorption. The continuous use of glucocorticoids is also related to pubertal delay and weight gain. These factors are associated with the incidence of fractures, osteoporosis, and the early loss of walking [8, 23–26]. The use of testosterone can lead to pubertal induction in DMD patients who use glucocorticoids in the long term and consequently improve bone mass accrual and bone healthy .
In G3, 25% of the participants used glucocorticoids. With the progression of the disease and muscle loss, the adverse effects on patient health caused by the chronic use of glucocorticoids may be more clinically relevant than are the potential benefits . G3 and G4 showed significantly lower BMD z-score values for both, lumbar spine and total body, than other groups differing by age (G1 and G2). This finding may be related to adolescence from the G2 ages to the G3 ages, when peak bone mass occurs in healthy patients .
The use of glucocorticoids inhibits the production of factors that regulate the hypothalamic-pituitary axis during puberty, leading to a decrease in growth hormone production, resulting in pubertal delay. Most participants in the age groups G1 and G2 use glucocorticoids, and pubertal delay caused by these drugs is one of the main concerns reported by DMD patients, leading to psychosocial and bone health consequences . In general, adolescents who do not have chronic diseases but have delayed sexual maturation have a reduced peak bone mass [29, 30].
Decreases in BMD in the lumbar spine and the total body are related to an increase in the risk of vertebral and long bone fractures, respectively, in DMD patients. Vertebral fractures cause spinal deformity, chronic pain, difficulty sitting, and decreased lung function, and long bone fractures may be associated with the permanent loss of ambulation .
According to a study by Ma et al. , approximately 20 to 60% of boys with DMD experienced low-trauma extremity fractures (usually of the femur or distal tibia), making it difficult or impossible for them to walk. Additionally, the risk of fractures increases as the DMD patients' survival increases . In agreement with the results of previous studies, the older patients in this study (G4) had a higher prevalence of reported bone fractures (33.3%) than other groups (Table 1), which may be associated with the total body BMD z-score being lower in this group (Table 2). Among these fractures, there is a predominance of long bone fractures. No patient reported a clinical history of vertebral fractures. Vertebral fractures can be asymptomatic in their early phases and thus undetected in the absence of monitoring . Among the four groups, G3 and G4 had the lowest percentages of participants who could walk independently and the lowest BMD z-scores, both in the lumbar spine and in the total body.
The early loss of ambulation caused by the progression of the disease gradually decreases the physical activity of these participants. The skeletal system has great modeling and remodeling dynamics, as it adjusts its properties in response to the load applied to it. Therefore, when there is a decrease in muscle use, bone loss occurs [9, 34]. Studies have shown that decreased muscle function and aging directly contribute to decreased bone density in patients with DMD , which is consistent with our results.
One of the strengths of our study is the sample size, which is larger than those of other studies that have also evaluated bone density in DMD patients [7, 8, 32]. Additionally, most studies have exclusively evaluated the lumbar spine or total body BMD, and in our study, BMD was evaluated in both areas.
The study has some limitations. As it is cross-sectional, it was impossible to analyze the cause-effects among glucocorticoids, walking ability, BMD, and disease progression. In addition, it is important to assess BMD associated with the assessment of bone fractures by imaging tests. Thus, longitudinal design studies should be conducted to assess the influence of these variables.
It is concluded that there is a moderate negative correlation between age and the BMD of the lumbar spine and a strong negative correlation between age and the BMD of the total body in the study population. Moreover, the older participants had a lower lumbar spine and total body BMD than younger ones. The results reinforce the relevance of assessing bone health in these patients at young ages to establish the most appropriate therapy and prevent bone fractures, as well as delay the early loss of walking and occurrence of fractures.