Fibroblast growth factor receptor-3 and estrogen receptor alpha as regulators of mandibular growth cessation in C5B7L male mice

INTRODUCTION

Orthognathic surgery is a treatment option for severe skeletal malocclusion, which often presents with a disfiguring appearance that can lead to psychosocial problems, particularly during adolescence. Surgery is recommended once the growth of the jaw has ceased to ensure more stable results and avoid the risk of requiring additional interventions in adulthood.^[1-5] Therefore, predicting the timing of mandibular growth cessation is essential. Several methods are used to predict the status of mandibular growth. One of the most popular methods is the cervical vertebrae method, which involves examining the morphology of C2, C3, and C4 on a cephalometric radiograph. However, this method is more sensitive for identifying patients during the peak of growth than at the end of growth.^[6]

The cessation of skeletal growth is marked by decreased osteogenesis and the fusion of the growth plate. Fibroblast growth factor receptor-3 (FGFR-3) has been identified as a negative regulator of bone growth. Loss-of-function of the FGFR-3 gene in mice exhibited a bone overgrowth phenotype.^[7,8] In humans, FGFR-3 gene mutations are associated with craniosynostosis in the Muenke and Crouzonodermoskeletal syndromes. Individuals affected with these diseases exhibit a prominent symphysis and narrow intergonial distance.^[9]

Growth plate fusion is influenced by estrogen. Estrogen exhibits a biphasic effect, stimulating bone growth at low concentrations during early puberty, and the concentration will continue to rise until it reaches a specific threshold, triggering the fusion of the growth plate. The molecular mechanism of these two opposing effects remains unknown, though it has been discovered that the maturation stage and estrogen levels influence the process.^[10-12] Estrogen requires receptors to perform its functions, and one of the most common receptors is estrogen receptor alpha (ER-α). Previous studies showed that the cartilage maturation process was impeded in mice with ER-α deficiency, as evidenced by an increase in cell proliferation and a decrease in hypertrophied chondrocytes.^[13] The presence of ER-α inactive mutations in humans has also been observed to result in elongated bodies and excessive growth due to the absence of fusion in the epiphyseal growth plate.^[14]

Mouse models have been widely used in studies on mandibular growth. Mice are known to have about 90% of the same genes, which is equivalent to 78.5% similarity in protein identity.^[15,16] Vora et al. (2016) investigated the postnatal craniofacial changes in C5B7L male mice and showed the mandibular growth peak at 7–28 days. Meanwhile, mice aged 56–84 days showed a reduction in mandibular growth. In addition, this study revealed a comparable growth pattern in the craniofacial region of both mice and humans.^[17] Although not entirely identical for humans, mice and rats remain the choice for bone growth research due to their ease of acquisition, breeding, and manipulation. This study aimed to examine the changes in FGFR-3 and ER-α levels in the condyle and plasma of C5B7L male mice during the growth period and the cessation of the mandibular growth period.

MATERIAL AND METHODS

The study was approved by the Animal Ethics Committee of the School of Veterinary Medicine and Biomedical Science (Ethics Approval No. 69/KEH/SKE/VII/2023) and conducted following the university’s guidelines for animal experimentation. This research was an observational study in C5B7L male mice. A total of 36 C5B7L male mice were categorized into three groups based on age: Group A (n = 12) for 28 days, Group B (n = 12) for 56 days, and Group C (n = 12) for 84 days. Sample size determinations were made using the resource equation approach for group comparison by Arifin and Zahiruddin (2017), with the equation: N = n × k, n = DF/kr + 1, where N = the total number of subjects, k = number of groups, n = number of subjects per group, and r = number of repeated measurements. The total sample size is = N × r if the animals must be sacrificed.^[18] Based on the calculation, it was found that the minimum number in each group was 12 mice. The mice were housed under the following environmental conditions: Room temperature of 19°C–23°C with 60–88% humidity, feed consumption of 10–18 g/body weight/day, and drinking ad libitum. Mice were anesthetized with ketamine and xylazine through the intraperitoneal route. Euthanasia was performed using the cervical dislocation technique. Then, blood plasma and condylar samples were collected. Enzyme-linked immunosorbent assay (ELISA) assays were conducted on all samples.

ELISA

The total protein was analyzed using the bicinchoninic acid (BCA) protein calorimetric assay kit (Elabscience E-BC- K318-M, Houston, Texas, USA) on protein extract and plasma samples. The FGFR-3 and ER-α concentrations in protein extract and plasma samples were assessed by ELISA tests using the Mouse FGFR-3 ELISA Kit (ELK4327, Denver, USA) and the Mouse ER-α ELISA Kit (ELK2932, Denver, USA).

RESULTS

The mean and standard deviation of FGFR-3 and ER-α in condyle and plasma for each age group are presented in [Tables 1 and 2]. It was observed that the concentrations of FGFR-3 and ER-α in condyle protein extracts had different patterns. The concentration of FGFR-3 in condyle declined as mice aged, but ER-α showed a reduction in 56-day-old mice and then increased again in 84-day-old mice. In plasma samples, the FGFR-3 level increased at age 56 days and reached its lowest at 84 days. In contrast, the concentration of ER-alpha were lowest at 28 days, showed a progressive increase with age, and reached the highest level at 84 days [Figures 1 and 2].

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Figure 1:

Fibroblast growth factor receptor-3 and estrogen receptor-alpha levels in condyle protein extracts. FGFR-3: Fibroblast growth factor receptor-3, ER-α: Estrogen receptor alpha

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Figure 2:

Fibroblast growth factor receptor-3 and estrogen receptor-alpha levels in plasma. FGFR-3: Fibroblast growth factor receptor-3, ER-α: Estrogen receptor alpha

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The ANOVA test indicated a statistically significant difference in mean values of FGFR-3 in both condyle and plasma, as well as in the concentration of ER-α in plasma, Kruskal–Wallis analysis revealed a statistically significant difference in the ER-α mean values in the condyle (P < 0.05) [Tables 1 and 2]. The Bonferroni test was used to analyze the inter-age group differences. Significant differences were observed in the levels of FGFR-3 in condyles of mice aged 28 and 56 days, as well as between 28 and 84 days. The Mann–Whitney test revealed similar results at the ER-α levels in the condyle. There was a significant difference in FGFR-3 levels in plasma between ages 56 and 84 days, whereas ER-α levels showed differences between ages 28 and 84 days [Table 3].

A Pearson correlation coefficient was performed to evaluate the relationship between the levels of FGFR-3 and mice age. A significant negative correlation existed between FGFR-3 levels in the condyle and mice age, as seen in [Table 4] (r = −0.733, P < 0.05). A moderate negative correlation was shown between ER-α levels and mice aged in the condyle. Meanwhile, there was no relationship in the plasma between the protein levels and the mice’s age.

DISCUSSION

This study aimed to investigate the age-related changes in FGFR-3 and ER-α levels in the condyle and plasma of the C5B7L male mice. The mandibular condylar cartilage (MCC) is distinct from other types of cartilage. MCC has a role as growth cartilage, characterized by hypertrophic cartilage, and as articular cartilage, which has fibrocartilage layers. During the growth phase, the condylar cartilage acts as growth cartilage, with four distinct zones: the fibrous layer, the proliferative layer, the mature cell layer, and the hypertrophic cell layer. The growth of the mandible results from three molecular processes: The proliferation of progenitor cells, cartilage matrix production, and hypertrophic chondrocyte enlargement. Among these three processes, the latter demonstrated the most significant influence. When reaching skeletal maturation, the condyle, which initially serves as a growth plate, transforms into articular cartilage. Histologically, it is marked by the disappearance of hypertrophic cartilage and is gradually substituted by fibrous cartilage.^[19,20]

The growth of the condyle is initiated by the proliferation and differentiation of progenitor cells into chondrocytes, regulated by Indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), SRY-box transcription factor 9 (Sox9). Chondrocytes then matured and differentiated into hypertrophic chondrocytes. At the beginning of the endochondral ossification process, the hypertrophic chondrocytes release collagen type X and express vascular endothelial growth factor (VEGF), which stimulates the infiltration of blood vessels, facilitating the migration of osteoprogenitor cells to the condyle. Osteoprogenitor cells then differentiate into osteoblasts and osteocytes, leading to osteogenesis. Once mandibular growth ceased, cell proliferation decreased, chondrocytes differentiated into osteoblasts at a higher rate, the extracellular matrix became compact, and the growth rate decreased.^[21,22]

Mice are commonly used in studies on mandibular growth due to their ease of acquisition and handling. Although mice and humans have different craniofacial morphology, the craniofacial growth pattern in mice is comparable to that in humans. Research using mice as models can provide valuable information on growth at the cellular, molecular, and genetic levels. Vora et al. discovered that the mandibula of C5B7L male mice had a growth spurt from days 7 to 28, and then the growth rate decreased. The mandibular length increment in 56 and 84-day-old C5B7L mice approached 0 mm/day, indicating that mandibular growth had ceased.^[17] Following this, Wei et al. also observed that the anteroposterior and vertical growth of the mandible was greatest between 7 and 21 days, followed by a minor increase through 120 days in female mice.^[23] Based on these findings, this study divided the age group into three categories: 28 days, marking the end of the mandibular growth spurt; 56 days, and 84 days, representing the range of mandibula growth cessation period.

FGFR-3 negatively regulates skeletal growth by inhibiting chondrocyte proliferation and differentiation. The FGFR-3 was broadly expressed from the immature cell layer to the hypertrophic layer in the 3-week-old rats, but it was absent from the hypertrophic layer at 8 weeks. It was indicated that FGFR-3 has an inhibitory effect in restraint of the proliferation and differentiation of chondrocytes. ^[24] In this study, the 28-day-old mice group had the highest concentration of FGFR-3 in the condyle, and then it declined in the 56-day-old and 84-day-old groups. This phenomenon might be due to the physiological role of FGFR-3 in suppressing chondrocyte proliferation and differentiation. FGFR-3 inhibits chondrocyte proliferation by down-regulating telomerase expression and reducing telomerase activity. Telomerase is essential for maintaining chondrocytes’ proliferative capacity and rate during bone growth.^[25] The cessation of growth is also marked by senescence, which is characterized by the loss of proliferative capacity in chondrocytes, and this process is induced by FGFR-3 signaling. In a cell line study, FGFR-3 inhibited chondrocyte proliferation by stimulating senescence. Senescence induced by FGFR-3 is characterized by the upregulation of senescence markers, including α-glucosidase, fibronectin, caveolin 1, lamin A, smooth muscle 22 alpha (SM22-A) and tissue inhibitor of metalloproteinases-1 (TIMP-1). FGFR-3 signaling activated the extracellular signal-regulated kinase (ERK) pathway and inhibited cell proliferation and growth arrest. The senescence or apoptosis induced by FGFR-3 likely depends on the intensity and duration of the signal.^[26]

The high levels of FGFR-3 at 28 days old might have caused the apoptosis of hypertrophic chondrocytes, which is essential for replacing cartilage with bone during ossification. The decrease in FGFR-3 levels at 56 and 84 days of age might be due to the reduced need for FGFR-3 signaling in the senescence process, as the growth process had already ceased. During this range of ages, the hypertrophic chondrocytes might have degenerated or differentiated into osteoblasts and begun the osteogenesis. The reduction of chondrocyte proliferation and degradation of hypertrophic chondrocytes leads to the cessation of mandibular growth.^[27]

Compared to the condyles, the concentration of FGFR-3 in plasma showed an increase in 56-day-old mice and a significant decrease in 84-day-old mice. These results could be because the level of FGFR-3 in the condyle demonstrated the roles of FGFR-3 as a local regulator of mandibular growth. Meanwhile, the levels of FGFR-3 in plasma reflected the roles of FGFR-3 in other organ systems. Besides its role in skeletal growth, FGFR-3 has a role in brain and lung development.^[28,29] This result also demonstrated that the level of FGFR-3 in plasma did not reflect what was happening inside the condyle. Estrogen plays a crucial role in the growth and development of the mandible. A systematic review by Omori et al. concluded that changes in estrogen levels, whether they increase or decrease, influence the dimensions of the maxilla and mandible.^[30] Furthermore, Küchler et al. (2021) demonstrated that estrogen deficiency increases mandibular length and condylar volume.^[31] Estrogen requires a receptor to perform its role, and the ER-α is the primary receptor involved. In a study conducted by Robinson et al. (2018), it was discovered that the administration of estrogen to ovariectomized mice reduced cell numbers in mandibular cartilage. Changes in the cell number might be due to a decrease in cell proliferation with estradiol treatment. This phenomenon was not found in mice with ER-α knockout (KO). These results suggested that ER-α is crucial for estrogen to inhibit mandibular condylar fibrocartilage growth.^[32]

In this study, the highest ER-α was observed in 28-day-old mice, which is assumed to be the end of the growth spurt period, followed by a decline in 56-day-old mice as the growth period had ceased. The high ER-α in 28-day-old mice might be due to the role of ER-α in reducing the proliferation of chondrocytes during the maturation of mandibular cartilage condyle), indicating the cessation of growth. Robinson et al. (2017) discovered that mice with ER-α deficiency condition showed increased chondrocyte proliferation and decreased hypertrophic chondrocytes, suggesting that ER-α has a role in suppressing chondrocyte proliferation.^[13] Another study by Yamada et al. showed that ER-α was expressed in all cartilage layers, but most prominently in the mature and hypertrophic chondrocyte layer.^[33] At the end of the mandibular growth peak, the hypertrophic chondrocytes diminished due to either the chondrocytes undergoing apoptosis or having differentiated into bone cells and undergone ossification.^[19,34] This might contribute to the decrease in ER-α levels in 56-day-old mice.

A significant increase in ER-α levels was observed in 84-dayold mice when growth was assumed to have stopped. This phenomenon might occur due to the role of ER-α in promoting senescence by regulating the expression of genes involved in chondrogenesis and osteogenesis. All the genes that were upregulated with estrogen in the mature MCC were modulated through ER-α indicating that this receptor is the primary mediator of estrogen-induced alterations in mature MCC. ER-α also plays a significant role in maintaining fibrocartilage homeostasis in the mandibular condyle. ER-α is involved in regulating various physiological processes, including chondrogenesis, matrix production, and protease activity, which are essential for temporomandibular joint health and its ability to withstand functional loads. ER-α is also essential for suppressing protease activity by enhancing the production of protease inhibitors, including protease inhibitor 15 and alpha-2-macroglobulin. Both inhibitors reduce matrix-metalloproteinase 9 activity, preserving the integrity of the cartilage.^[32] These physiological processes might cause elevated ER-α in 84-day-old mice.

The plasma concentration of ER-α had a distinct trend in comparison to the condyle. In plasma, the concentration of ER-α increased with age. This could be due to the roles of ER-α in various organ systems, including reproduction, brain, cardiovascular, and skeletal systems. Mice with ER-α KO have infertility due to the inability of the uterus to receive estrogen. Estrogen and its receptors are also known to play a crucial role in regulating cardiovascular metabolism, reducing cardiomyocyte apoptosis, and regeneration. In brain development, estrogen is known to play a significant role, particularly in sexual dimorphism.^[35]

CONCLUSION

This study showed that the levels of FGFR-3 and ER-α in condyle and plasma varied with age during the growth and end of the mandibular growth period. The maximum levels of FGFR-3 and ER-α in the condyle were seen in 28-dayold C5B7L male mice, followed by a significant reduction, indicating FGFR-3 and ER-α roles in the cessation of mandibular growth. The level of FGFR-3 in plasma increased and reached the highest level at 56-day-old, then decreased at 84-day-old. Meanwhile, ER-α concentration in plasma was increased with age. The limitation of this study was the absence of mandibular length measurement in mice, which precludes the assessment of mandibular size at different ages. Future studies are needed to accurately depict the role of FGFR-3 and ER-α at the end of mandibular growth.