Effects of methylphenidate on femoral bone growth in male rats

Abstract

The use of Methylphenidate (MP) can have adverse effects on bone growth and mineralization. This study aimed to investigate the underlying pathophysiology of MP-induced skeletal deficits in growing rats using stereological and immunohistochemical methods. Male rats, aged 4 weeks, were orally treated with MP through an 8-h/day water drinking protocol. The rats (n=30) were randomly divided into three groups: MP-High Dose (30/60 mg/kg/day MP), MP-Low Dose (4/10 mg/kg/day MP), and control (water only). After 13 weeks, the femoral bones were assessed using calliper measurements, dual-energy X-ray absorptiometry, and biomechanical evaluation. The total femur volume, cartilage volume, growth zone volume, and volume fractions were determined using the Cavalieri method. Immunohistochemical analyses were conducted using alkaline phosphatase and anti-calpain antibody staining. Rats exposed to MP exhibited significant reductions in weight gain, femoral growth, bone mineralization, and biomechanical integrity compared to the control group. The total femoral volume of MP-treated rats was significantly lower than that of the control group. The MP-High Dose group showed significantly higher ratios of total cartilage volume/total femoral volume and total growth zone volume/total femoral volume than the other groups. Immunohistochemical evaluation of the growth plate revealed reduced osteoblastic activity and decreased intracellular calcium deposition with chronic MP exposure. The possible mechanism of MP-induced skeletal growth retardation may involve the inhibition of intracellular calcium deposition in chondrocytes of the hypertrophic zone in the growth plate. In this way, MP may hinder the differentiation of cartilage tissue from bone tissue, resulting in reduced bone growth and mineralization.

Introduction

Attention Deficit Hyperactivity Disorder (ADHD), which is one of the most commonly seen psychiatric disorders in children, is a developmental disorder characterized by a short attention span, distractibility, forgetfulness, hyperactivity, excessive talking and impulsiveness. ADHD can lead to problems such as academic failure, behavioral problems, problems in family and peer relationships, sleep disorders, substance abuse and criminal behavior. The frequency of ADHD in children has been reported to be 5%–10%. ¹ Pharmacotherapy is the most effective treatment option for ADHD treatment. ² Methylphenidate (MP), a central nervous system stimulant, is one of the first-choice drugs for ADHD treatment in children and adolescents. ³ The efficacy of MP on ADHD symptoms is 75%–80% and it is well tolerated in children and adolescents. ⁴

ADHD requires long-term treatment that may continue throughout the entire education life of the child. Weight and height gain deficits are the adverse effects of MP that concern physicians and families. ⁴ Several clinical studies have shown that MP treatment negatively affects weight gain and height growth in children and adolescents.^5–7 Similar findings have also been reported in preclinical animal studies investigating the skeletal effects of MP.^8–10 An experimental study revealed that chronic MP treatment resulted in dose-dependent growth suppression, decreased bone mineral density (BMD) and reduced bone strength in growing rats. The MP-induced skeletal effects were then seen to recover rapidly following treatment cessation. ⁸

There have been few studies investigating the pathophysiology of the adverse effects of MP on bone growth and bone quality. In an animal study that histologically examined the adverse effects of MP on the skeleton, it was observed that MP increased osteoclast activity and bone resorption in male rats. ¹¹ Another study revealed that MP adversely affected new bone formation in the premaxillary suture by decreasing osteoblastic activity in rats. ¹²

Upon reviewing existing literature, it becomes evident that while the inhibitory effect of MP on bone growth and mineralization is recognized, there is a notable absence of studies focusing on the immunohistochemical exploration of cellular attributes and mineralization within the growth plate. Furthermore, there appears to be a lack of investigations that quantitatively assess the volume of the growth plate and femoral bone.

The aim of this study was to examine the potential effects of long-term MP exposure on bone growth, BMD and bone biomechanical integrity in developing rats. It was also aimed to investigate the underlying pathophysiology of MP-induced adverse effects on the skeleton using stereological and histological methods.

We hypothesized that (1) Chronic MP exposure might lead to reduction in growth, mineralization and stiffness of femoral bones in juvenile rats. (2) MP treatment might affect growth plate and femoral bone volume due to its inhibitory effects on bone growth and mineralization pathways. (3) Immunohistochemical analysis of the growth plate in the MP treated rats could reveal altered expression patterns of key markers associated with growth and mineralization. (4) It is possible that MP’s interference with growth plate function could result in disrupted endochondral ossification and subsequently affect bone elongation.

Materials and methods

Permission for this study was obtained from the Local Ethics Committee (decision no: B.30.2.ODM.0.20.09.00-050.04) and the experimental procedures were performed in Ondokuz Mayis University Center for Experimental Animal Application and Research. The study was conducted in accordance with the ARRIVE guidelines.

Animal study

The study involved a total of 30 male Wistar-Albino rats, aged 4 weeks and weighing between 76 and 106 g. Male rats were used because it has been reported that the side-effects of MP on the bone are more pronounced in male rats compared to female rats. ¹¹ The groups and rats were numbered and kept in separate cages at standard room temperature with a 12-h light-dark cycle. Standard rat food was provided. The rats were randomly separated into three groups of 10: Control group (group given water only), Low dose Methylphenidate group (MP-Low Dose), and High dose Methylphenidate group (MP-High Dose). The MP treatment was given through drinking water for 8 h daily. The MP-Low Dose group was given 4 mg/kg dose in the first hour and 10 mg/kg dose in the following 7 h, dissolved in distilled water. The MP-High Dose group was given 30 mg/kg dose in the first hour and 60 mg/kg dose in the following 7 h, dissolved in distilled water. The control group rats were given water only for 8 h daily. No water was given to the rats during the remaining 16 h of the day. In this study, the administration protocol of the medication has been implemented to mimic its usage and pharmacokinetic profile in humans based on previous studies. The administration of MP in rats through injection or gavage results in a rapid and high-dose plasma level, not reflecting the usage in humans. Extended-release MP, when taken orally in children and adolescents, maintains its effect throughout the school time due to its slow-release formulation. Oral administration of extended-release MP plasma concentration increases rapidly reaching an initial maximum at about 1 h, followed by gradual ascending concentrations over the next 5 to 9 h after which a gradual decrease begins. Thanos et al. ¹³ reported that, this administration protocol of MP reflected the use of extended-release MP in humans by measuring drug plasma levels in rats. This dosage protocol has been reported to be similar to the pharmacokinetic profile of MP in children and has been extensively used in several previous studies.^11,13,14 The treatment was applied for 13 weeks. In rats, the age between 4 and 8 weeks corresponds to the period of preadolescence. ¹⁵ The 13 weeks of MP use in 4-week-old rats represents chronic MP use that begins in childhood and continues throughout the educational life in humans. During the study, the weight of the rats was measured monthly. One rat died in the MP-High Dose group during the study. After 13 weeks, all the rats were euthanised, and the bilateral femurs were removed from the muscle tissue. The femurs were placed in physiological saline and stored at −20°C until analysis. The left femurs were used for the evaluation of the bone length, bone density and biomechanical integrity, while the right femoral bones were used for histological examination and stereological examination. The left femoral bones were thawed to room temperature before the evaluations, and then re-frozen for use in the next step.

Measurements with calipers

The bone dimensions were measured using digital calipers on the left femur of the rats in each group. The femur length measurements were taken by adjusting the calipers with one end on the head of the femur and the other on the medial condyle. The anteroposterior diameter and mediolateral diameter were measured from the centerline of the femur diaphysis.

Dual-energy X-ray absorptiometry (DEXA) assessment

After measuring the left femurs of the rats with the calipers, each was evaluated separately using the DEXA method (DEXA QDR-4500 Hologic, Bedford, MA, USA). BMD and bone mineral content (BMC) values were measured for each femur.

Biomechanical assessment

The proximal and distal ends of the femurs of the rats were secured to a specially made apparatus, and the anterior femur surface was placed in a three-point bending device in such a way that it faced upwards (Gunt WP300, Hamburg, Germany). Force was applied to the middle section of the femur with a 5 mm/min crosshead feed rate until a fracture formed on the surface. Fracture formation was monitored by the device’s own software and detected by the instantaneous decrease observed after the maximum load was reached. The applied load and the resulting displacement were instantly displayed in the software. The stiffness values were calculated over the ultimate displacement and force data.

Histological assessment

Tissue processing, blocking, sectioning, and staining

The femoral specimens of all the groups were fixed in 10% formalin for 1 week, then the tissues were decalcified in a 5% formic acid solution at room temperature for 21 days. At the end of the decalcification process, the tissues were washed under tap water, and the dehydration process was performed using graded alcohol series. The samples were then cleared with xylene and embedded in fresh paraffin. For stereological and light microscopic evaluations, 10 µm thick coronal serial sections were cut from the paraffin tissue blocks with a 100 µm sampling interval according to the systematic random sampling rule. The sections were stained with Hematoxylin-eosin.

Immunohistochemical analysis

For the immunohistochemical staining, 5 μm sections were cut from each tissue block and stained using alkaline phosphatase and anti-calpain antibodies, respectively. Antibodies were detected with AEC (Invitrogen, USA), counterstained with Mayer’s hematoxylin (Merck, USA), dehydrated and then mounted with permanent media.

Immunolabelling of sections with antibodies was scored semi-quantitatively. A randomly selected intense area in points of positively labeled cells was selected, and the intensity of reactivity was evaluated at ×100 final magnification using an Olympus BX43 F (Tokyo, Japan) microscope. The grading of stained sections was scored as 0 if there were no stained cells; as + 1 if < 25% of cells were stained; +2 if 25% to <50% of cells were stained; +3 if 50%–75% of cells were stained; and +4 if ≥75% of cells were stained.

Stereological evaluation

The total femur volume, cartilage volume, and growth zone volume were calculated quantitatively using the Cavalieri method in the sections taken according to the systematic random sampling rule. A stereological grid was placed on the sections, and the points that superimposed with tissue were counted. This method allows the researcher to calculate the surface area [a(p)], and then, the volume of the structure is obtained as a result of the surface area multiplied by the section thickness. The area (A) of the cross-section can be calculated by multiplying the total number of points (Σp) and the area represented by a point [a(p)]. ¹⁶

$\mathrm{A}=\sum p\hspace{0.17em}\times \hspace{0.17em}\left[a\left(p\right)\right]$

Volumetric measurements were performed by considering the appropriate coefficient of error (<0.05) and coefficient of variation (<0.20) values determined after the pilot study. Volume fractions in percentages were calculated by dividing the total growth zone volume and cartilage volume by the total femoral volume (total growth zone volume/total femoral volume and total cartilage volume/total femoral volume, respectively) and multiplying the data by 100.

Statistical analysis

Statistical analyses of the data were performed using SPSS 21.0 for Mac software (IBM Corporation). The One Way ANOVA (Tukey Post-Hoc Test) test was used to compare the groups. The level of statistical significance was set at 0.05 (p).

Results

Weight gain

The weight gain was reduced during the study period depending on the dose of MP applied to the rats and compared to the control group (Figure 1).

Figure 1.

The mean weight values of the groups at baseline, and 1, 2, and 3 months. The mean values of the groups were compared using the One Way ANOVA test. Pair wise comparisons were performed using Tukey Post-Hoc Test. A value of p<.05 was considered statistically significant. Significant differences between the groups were shown with different symbols (*,#,&) in the figure.

At the end of the first month, the mean weight of the MP-Low Dose group was 12% less than that of the control group. The mean weight of the MP-High Dose group was 18% less than that of the control group. At the end of the second month, the mean weight of the MP-Low Dose group was 8% less than that of the control group, and the mean weight of the MP-High Dose group was 14% less than that of the control group. At the end of the third month, the mean weight of the MP-Low Dose group was 4% less than that of the control group, and the mean weight of the MP-High Dose group was 12% less than that of the control group. With the exception of the difference between the control group and MP-Low Dose group at the end of the third month, all the other differences were statistically significant.

Bone dimensions

Bone size measurements made using a digital caliper showed that the lengths of the femurs of the MP group rats were shorter and the diameters were thinner in a dose-dependent manner (Figure 2).

Figure 2.

Measurements of femurs with digital calipers. (a) comparisons of femur lengths; (b) comparisons of anterior-posterior (AP) diameters; (c) comparisons of medio-lateral (ML) diameters of the groups. The measurements of the three groups were compared using the One Way ANOVA test. Pairwise comparisons were performed using the Tukey Post-Hoc Test. A value of p<.05 was considered statistically significant. Significant differences between groups were shown with asterisks (*) in the figure.

The femur lengths of rats given MP were 1% shorter at the low dose and 3% shorter at the high dose compared to the control group. The difference in mean values of femur length was found to be statistically significant between the control group and the MP-High Dose group. The AP diameter of the femurs of rats given MP was 5% narrower at the low dose and 7% narrower at the high dose compared to the control group. The difference in mean values of AP diameter was found to be statistically significant between the control group and the MP-High Dose group. In the ML measurement, the MP-Low Dose group was 5% narrower and the MP-High Dose group was 7% narrower than the control group. This difference was statistically significant in both groups compared to the control group.

Densitometric evaluation

According to the results of the densitometric evaluation, the average BMD and BMC values of the femurs in the rats treated with MP were found to be lower (Figure 3).

Figure 3.

Results of the femoral densitometry assessment. (a) comparison of femoral BMC values; (b) comparison of BMD values of the groups. The mean values of the three groups were compared using the One Way ANOVA test. Pairwise comparisons were performed using the Tukey Post-Hoc Test. A value of p<.05 was considered statistically significant. Significant differences between groups were shown with asterisks (*) in the figure.

The mean BMC value of the MP-Low Dose group was 10% lower than that of the control group, and the mean BMC value of the MP-High Dose group was 15% lower than that of the control group. The mean BMD value of the MP-Low Dose group was 5% lower than that of the control group, and the mean BMD value of the MP-High Dose group was 7.5% lower than that of the control group. These differences were statistically significant.

Biomechanical testing

The biomechanical tests on the femurs showed that the mean Ultimate Force and Stiffness values of the MP-Low Dose and MP-High Dose groups were significantly lower than those of the control group (Figure 4).

Figure 4.

Results of the biomechanical testing. (a) comparison of femoral ultimate force values; (b) comparison of femoral stiffness values. The mean values of the three groups were compared using the One-Way ANOVA test. Pairwise comparisons were performed using the Tukey Post-Hoc Test. A value of p<.05 was considered statistically significant. Significant differences between the groups were shown with asterisks (*) in the figure.

The mean Ultimate Force of the MP-Low Dose group was 36% lower, and the MP-High Dose group was 48% lower than that of the control group. The mean Stiffness value of the MP-Low Dose group was 32% lower, and the MP-High Dose group was 52% lower than that of the control group.

Histological evaluation

Stereological evaluation revealed that the total femoral volume values of MP-exposed rats were significantly lower than those of the control group, with a 37% reduction in the MP-Low Dose group and a 48% reduction in the MP-High Dose group (Figure 5).

Figure 5.

Representative light microscopical images (HE staining) of the relevant groups and the stereological results were shown (a). In the HE stained sections of the Control, MP-Low Dose and MP-High Dose groups, the growth zone in the Control and MP-High Dose groups was observed more intensely compared to the MP-Low Dose group. In addition, the increase in cartilage volume in the Control and MP-High Dose groups compared to the MP-Low Dose group was noticed. Density of the new bone area and osteoblastic activity were also more clearly observed in the MP-High Dose group at high power magnification image (&). Results of stereological evaluation (b) comparison of total femoral volume, total cartilage volume and total growth zone volume. The results were compared between the groups within each dosing protocol using One Way ANOVA, followed by pairwise comparisons with the Tukey Post-Hoc Test. A value of p<.05 was considered statistically significant. Significant differences between the groups were shown with asterisks (*) in the figure.

The total cartilage volume and growth zone volume of the MP-Low Dose group were significantly lower than those of the control and MP-High Dose groups. The MP-High Dose group was similar to the control group regarding these variables (Figure 5).

The significantly highest growth zone volume fraction, expressed as a percentage of the total femoral volume, was determined in the MP-High Dose group (Figures 6 and 7). The cartilage volume fraction of MP-High Dose group was also significantly higher than that of the MP-Low Dose and control groups (Figure 6).

Figure 6.

Percentages of volume fractions of the groups. (a) The ratio of total growth zone volume to total femoral volume as a percentage; (b) The ratio of total cartilage volume to total femoral volume. The results were compared between the groups within each dosing protocol using One Way ANOVA, followed by pairwise comparisons with the Tukey Post-Hoc Test. A value of p<.05 was considered statistically significant. Significant differences between the groups were shown with asterisks (*) in the figure.

Figure 7.

Schematic comparison of the groups in terms of femoral length (FL), mediolateral diameter (ML) and total growth zone volume/total femoral volume (%).

The results of the study were summarized in the Table 1.

Table 1.

Outcomes of the study.

	Control (Mean ± SD)	MP-low dose (Mean ± SD)	MP-high dose (Mean ± SD)	p-value
Body weight (gr)
Baseline	91.5 ± 7.1	93.5 ± 6.4	91 ± 6.6	0.697
First month	229 ± 10.4	200.4 ± 13.9	187.3 ± 9	0.000
Second month	261 ± 12	240 ± 16.2	223.6 ± 10.5	0.000
Third month	292.8 ± 6.6	280.4 ± 14	256.9 ± 22	0.000
Bone size (mm)
Femur length	33.81 ± 0.94	33.26 ± 0.37	32.76 ± 0.81	0.02
AP diameter	3.08 ± 0.11	2.92 ± 0.28	2.86 ± 0.97	0.05
ML diameter	4.1 ± 0.16	3.89 ± 0.11	3.81 ± 0.14	0.001
Densitometry
BMC (g)	0.38 ± 0.01	0.34 ± 0.01	0.32 ± 0.02	0.000
BMD (g/cm2)	0.211 ± 0.01	0.200 ± 0.01	0.195 ± 0.01	0.001
Biomechanics
Ultimate force (N)	127.7 ± 21	81 ± 26.9	65.3 ± 30.5	0.000
Stiffness (N/mm)	102.4 ± 39.7	69.3 ± 17.9	48.7 ± 19.8	0.001
Stereology (µm3)
Femur volume	3.32 ± 0.27	2.08 ± 0.07	1.72 ± 0.07	0.000
Cartilage volume	1.12 ± 0.16	0.88 ± 0.08	1.11 ± 0.02	0.012
Growth zone volume	0.06 ± 0.001	0.04 ± 0.01	0.06 ± 0.003	0.000
Volume fractions (%)
Growth Zone/Total femur	1.8	2.02	3.9	0.000
Cartilage/Total femur	3.3	4.2	6.5	0.000

Immunohistochemical results

The immunohistochemical results were shown in Figures 8 and 9.

Figure 8.

Immunohistochemical scoring of the relevant markers of the groups shown as percentages.

Figure 9.

Sections from the Control, MP-Low Dose and MP-High Dose groups were immunohistochemically stained with alkaline phosphatase antibodies (a) and anti-calpain antibodies (b). Black arrows indicate alkaline phosphatase (+) (a) and anti-calpain (+) cells (b). In the Control and MP-High Dose group, alkaline phosphatase (+) cells were more common (scores +++ and ++, respectively) than in the MP-Low Dose group (score +). Thus, higher levels of osteoblastic activity were observed in the Control group and the MP-High Dose group compared to the MP-Low Dose group. In the Control and MP-Low Dose groups, anti-calpain (+) cells were more common (scores ++ and ++, respectively) than in the MP-High Dose group (score +). Thus, intracellular calcium increase in the MP-High Dose group was observed to be lower than in the MP-Low Dose and Control groups (Mayer Hematoxylin, Lillie’s modification).

Discussion

The hypothesis of this study was that long-term use of MP in young, growing rats would have adverse effects on weight gain, bone growth, bone mineralization, bone biomechanical integrity and histomorphological structure. Total femur, growth zone, and cartilage volumes were assessed using stereological methods and volume fractions were also calculated. The MP-induced cellular changes in the femoral bone were investigated through immunohistochemical evaluation.

Numerous clinical and experimental studies have demonstrated that MP decreases weight gain in a dose-dependent manner. One of the most comprehensive studies on MP, the MTA study, found that after 36 months of monitoring, children using MP were, on average, 2.71 kg lighter compared to control children who did not use the drug. ¹⁷ In a meta-analysis of studies based solely on Z-score-based height/weight monitoring, it was reported that MP prevented weight gain in a dose-dependent manner, especially during the first 6 months of treatment. ⁶ Animal experiments have also shown that MP inhibits weight gain, especially at high doses.^11,14,18 Even when the pair-feeding paradigm was applied in one study, weight gain decreased in the high-dose MP group. ¹¹ Similarly, the results of the current study showed that MP significantly inhibited weight gain in rats in a dose-dependent manner. The negative effect of MP on weight gain was significant in the MP-Low Dose group in the first month, but the difference between that and the control group disappeared in the 3rd month. This finding may be due to the fact that side-effects are initially very intense and gradually diminish over time as tolerance to MP develops.

It is known that MP has a negatively effect on height growth. The MTA study reported that after 36 months of follow-up, MP caused an average 3.04 cm reduction in height compared to non-medicated control subjects, and this difference continued during adolescence and adulthood. ¹⁷ In a study by Komatsu et al., ¹⁴ a significant reduction was observed only in the anterior-posterior diameter of the femoral bone. The current study results showed that MP caused a significant decrease in the femur length and anterior-posterior diameter in the MP-High Dose group, and compared to the control group, a significant difference was detected in the mediolateral diameter in both the MP-Low Dose and MP-High Dose groups.

The study results showed that at both low and high doses, MP caused a decrease in bone mineralization and density compared to the control group. The results of previous studies investigating the effects of MP on bone metabolism in children have been inconsistent. In a pilot study by Lahat et al., no significant difference in BMD was found between 10 children treated with MP for 1–2 years and control subjects. ¹⁹ A cross-sectional study of a community sample revealed that children and adolescents using stimulants had lower bone mass than those who did not. ²⁰ Poulton et al. found that dextroamphetamine and MP treatments caused lower BMD in a 3-year follow-up study.^21,22 A recent study on a nationally representative adult sample revealed that stimulant ADHD medication usage was associated with decreased BMD in the skull and thoracic spine. ²³ Another study of juvenile rats reported that high-dose MP caused a significant decrease in BMC and BMD, but these parameters were not significantly different following a recovery protocol. ¹⁴ Various mechanisms have been proposed to explain the reduction in bone density and mineralization caused by stimulants. One of these mechanisms may be that calcium and vitamin D are deficient as a result of the MP effect on reducing appetite and weight gain. Another possible mechanism could be that increased sympathetic activity may decrease bone mass by reducing osteoblastic activity and increasing osteoclastic activity. ²⁴

The results of the current study biomechanical assessment demonstrated that bone stiffness decreased and the bone became more fragile, supporting the other findings of the study. While there are publications that have stated that MP reduced the risk of fracture, there are also others stating that it increased the risk of stress fractures.^25–28 In children with ADHD, symptoms such as hyperactivity, impulsiveness, and inattentiveness are known to lead to traumatic injuries, and it has been shown that treatments for ADHD (stimulant and non-stimulant) reduce these symptoms and thereby, the risk of traumatic fractures. ²⁶ Stress fractures occur as a result of repetitive micro-trauma to the bone. ²⁸ Large-scale studies on soldiers have shown that using MP significantly increased the risk of stress fractures. ²⁸ In a rat study, high doses of MP reduced the ultimate force of the femur by 20% and energy to failure by 33%. ¹⁴ In another experimental study, it was observed that only male rats exposed to high doses of MP had a biomechanically weaker femoral bone. ¹¹

In the current study, total femur volume, growth zone volume, and cartilage volume and volume fractions were also evaluated to shed light on the adverse effects of MP on bone growth. Total femur volume was significantly lower in the MP-exposed rats compared to the control group, and the growth zone and total cartilage volumes were significantly lower in the MP-Low Dose group compared to the other groups. In contrast, the MP-High Dose group was similar to the control group in terms of these variables.

The analyses of the volume fractions in this study revealed some striking results. The ratios of growth zone volume/total femoral volume and cartilage volume/total femoral volume were significantly higher in the MP-High Dose group compared to the other groups. This finding suggested that high doses of MP cause the total femur volume to be smaller, while the growth zone and cartilage volumes remain larger compared to the femur volume. This indicated that high doses of MP have a negative impact on bone formation and growth by inhibiting the transformation of cartilage tissue into bone tissue in the growth zone.

The immunohistochemical staining results of this study revealed interesting findings. In the control and MP-High Dose group specimens, ALP (+) cells were more common (scores +++ and ++, respectively) than MP-Low Dose group (score +). Thus, it was observed that levels of osteoblastic activity were higher in the control group and MP-High Dose group compared to the MP-Low Dose group. Endochondral ossification is the process of bone formation and growth in which cartilage scaffolds are gradually replaced by bone. During endochondral ossification, chondroblasts differentiate into chondrocytes and begin to secrete ALP, an enzyme that acts as a nucleator for depositing minerals on the template. ALP plays an important role in osteoid formation and mineralization. ²⁹ This result obtained in the study suggested that in rats exposed to high-dose MP, more ALP may be synthesized to facilitate the conversion of chondrocytes into osteocytes in the hypertrophic zone of the growth plate and the mineralization of bone. To the best of our knowledge, there are no studies in the literature examining bone ALP levels related to MP exposure. In the study by Komatsu et al., ¹⁴ although bone growth and mineralization decreased, the serum ALP level was unexpectedly higher in the high-dose MP group. There is a need for further studies to examine bone and serum ALP concomitantly.

The density of anti-calpain (+) cells was observed to be lower in the MP-High Dose group compared to the control and MP-Low Dose groups. Calcification of cartilage occurring in the lower segment of the hypertrophic zone within the growth plate plays a crucial role in the normal elongation of long bones. Calpain is a proteolytic enzyme which has been identified in the lower hypertrophic zone of the growth cartilage. This enzyme has garnered considerable attention due to its potential role as a proteinase that propels the calcification process within the growth cartilage. The precise localization of calpain within chondrocytes and the matrix of the hypertrophic zone, strongly indicate a significant role for this enzyme in the process of matrix calcification that occurs within the growth cartilage zones. ³⁰ In this study, immunohistochemical assessment with anti-calpain was performed to evaluate the impact of MP on the calcification of the hypertrophic zone and the ossification of cartilage. The results of this study support each other, and when interpreted together, shed light on the negative effects of MP on bone growth. These findings suggest that MP may block intracellular calcium accumulation in the cells of the hypertrophic zone of the growth plate, thereby inhibiting differentiation of chondroblasts to osteocytes and suppressing bone formation and growth.

As far as is known, there have been few studies examining the adverse effects of MP on bone histology. Uddin et al. reported that MP exposure resulted in a dose- and sex-dependent increase in osteoclast activity in juvenile male rats. ¹¹ Despite the increased physical activity in MP-treated rats, increased osteoblast activity was not observed.^11,14 Another study examined the effects of MP on bone healing following rapid maxillary expansion in rats. MP was seen to cause suppression of new bone formation, and decrease the number of osteoblasts and capillaries in the premaxillary suture. No statistically significant difference was observed between the MP-exposed rats and the control group in respect of the number of osteoclasts. ¹²

MP-induced deficiencies in bone growth, mineralization, and strength may be based on the direct effects of MP on bone as well as the reduced weight gain. An in vitro study provides evidence supporting the direct impact of MP on bone tissue. ¹¹ Further studies are needed to understand the exact mechanism of MP-induced skeletal growth retardation.

There were some limitations to this study, primarily that it was an experimental study on rats, so the findings may not correspond to the clinical usage of MP in children and adolescents. Second, only male rats were included, so gender-dependent effects of MP were not investigated. Third, only the femoral bone, which grows with endochondral ossification, was evaluated, and other bones, which grow with intramembranous ossification were not examined. In addition, no analysis was made of the serum ALP level.

In conclusion, this study found that chronic MP treatment in juvenile rats led to dose-dependent deficiencies in femoral growth and integrity, resulting in weaker and less mineralized bones. Both low and high doses of MP significantly reduced total femur volume. The high-dose MP group had higher volume fractions, indicating inhibition of cartilage-to-bone differentiation and impaired bone growth. MP treatment also affected bone ALP activity and reduced anti-calpain positive cell density. These findings collectively suggest that MP, especially at high doses, inhibits calcium accumulation in growth zone cells, hindering the differentiation of chondrocytes into osteoblasts and impeding bone growth.

ADHD is a neurodevelopmental disorder, which may require treatment for many years. High rates of comorbidity with disruptive behaviors and anxiety disorders are seen in ADHD, which requires the combination of MP with antipsychotics (AP) and selective serotonin reuptake inhibitors (SSRI). ³¹ Evidence has shown that APs and SSRIs also negatively affect bone metabolism.^18,20,32 Therefore, when prescribing MP, it is crucial to avoid off-label use, administer the minimum effective dose, carefully plan polypharmacy, monitor side-effects closely, and consider drug holidays when appropriate.