The effects of dietary loading on the transdifferentiation of condylar chondrocytes

Abstract

Introduction:

Transdifferentiation of chondrocytes into bone cells explains most condylar growth during prenatal and early postnatal stages, but the mechanisms regulating chondrocyte transdifferentiation during late postnatal growth remain unknown. This study aimed to quantify the effects of dietary loading on chondrocyte-derived osteogenesis during late postnatal condylar growth.

Methods:

Two compound mouse lines were used to trace the fate of chondrocyte lineage in vivo. Twelve 3-week-old male Aggrecan-Cre^ERT2 (Acan^Lineage); R26R^TdTomato; 2.3 Col10a1-GFP and twelve 3-week-old male Col10a1-Cre (Col10a1^Lineage); R26R^TdTomato; 2.3Col1a1-GFP were randomly divided into experimental (soft-food diet, n = 6) and control (hard-food diet, n = 6) groups and kept for 6 weeks. One time, tamoxifen injections were given to Acan^Lineage mice at 3 weeks. Radiographic, microcomputed tomographic, and histomorphometric analyses were performed.

Results:

Radiologic analysis showed that mice with a soft-food diet had smaller mandible lengths as well as decreased bone volume and density for their condylar process. Histologically, mice with soft diets had reduced activity in chondrocyte proliferation and maturation compared with the controls. Cell lineage tracing results showed the number of Acan^Lineage-derived bone cells (293.8 ± 39.8 vs 207.1 ± 44.6; P = 0.005), as well as total bone cells (445.6 ± 31.7 vs 360.7 ± 46.9; P = 0.004), was significantly higher in the hard-diet group than in the soft-diet group, whereas the number of non-Acan^Lineage-derived bone cells was not significantly different among groups (P = 0.938). Col10a1^Lineage mice showed the same trend.

Conclusions:

Dietary loading directly affects condyle chondrogenesis and chondrocyte transdifferentiation, which alters the extent of condylar growth and remodeling.

Orthodontists need to better understand how and why the mandible adapts to environmental changes, which is essential for orthopedic intervention and its treatment effects. A decrease in masticatory strength because of softer diets has been suggested as a major culprit behind phenotypic mandibular variability.¹ Hyperdivergent features observed in human patients, such as increased mandibular plane angles, gonial angles, and anterior face heights, have been directly linked to weaker masticatory muscles.^2–5 Conversely, when exercises to strengthen the masticatory muscles are completed, the mandible adapts and illustrates phenotypic changes opposite to those of hyperdivergent subjects.⁶ These features have also been demonstrated in many animal studies by manipulating their diet.^7–9 The subsequent changes in quality, size, and shape of cartilage and bone after the alteration of diet have led to the conclusion that dietary loading plays a significant role in the development of the craniofacial complex.¹⁰

One reason for the adaptability of the mandible is the temporomandibular joint, specifically the mandibular condyle. It is composed of bone and a layer of secondary cartilage on its superior surface. This type of cartilage differs from primary cartilage in that it requires mechanical loading for its growth and reacts to stimuli for its maintenance.^11–13 There is a clear association between soft diet and condylar growth. The studies on rodents have found decreased cartilage thickness along with reduced length and bone volume of the condylar process in the soft-diet group.^7–9 However, it is unknown how cellular changes translate to these macroscopic changes and whether the adaptation of condylar cartilage to mechanical stimuli directly links to the reduction of bone in the condylar process.

Temporomandibular joint condyles grow via endochondral bone formation, beginning with mesenchymal cells that proliferate and differentiate into chondrocytes. As the cartilage grows, chondrocytes become entrapped and enter a hypertrophic state. It has been traditionally thought that the hypertrophic chondrocytes undergo apoptosis, followed by an invasion of cells from the underlying bone marrow that erode the calcified cartilage and initiate angiogenesis, bringing in cells that deposit new bone.¹⁴ Using cell lineage tracing, a powerful tool to trace cell fate in vivo, Jing et al¹⁵ found that up to 80% of bone cells in the condylar process arise via direct transdifferentiation of chondrocytes during prenatal and early postnatal development. This is also consistent with the results obtained from fetal and postnatal metaphyseal growth.^16–22 Together, these studies demonstrated that chondrocytes play a critical role in endochondral bone formation and that chondrogenesis and osteogenesis are 1 continuous process. However, it is still unknown to what extent the condylar chondrocytes contribute to late postnatal condylar growth and whether dietary loading has a direct role in regulating condylar chondrocyte transdifferentiation.

This study aimed to determine (1) the role and contribution of chondrocyte-derived osteogenesis occurring during late postnatal condylar growth and (2) whether dietary loading affects the condylar bone by regulating the chondrocyte transdifferentiation. It was hypothesized that a consistent decrease in dietary loading would reduce chondrocyte differentiation and transdifferentiation, resulting in mandibular growth changes. Two Cre lines were used to trace the fate of chondrocytes: Aggrecan-Cre^ERT2 was used to trace all chondrocytes and their descendants after tamoxifen administration at 3 weeks; Col10a1-Cre was used to trace hypertrophic chondrocytes and their descendants constitutively once its expression began around E14.5.²³ The expression of 2.3Col10a1-GFP (a marker for osteoblasts and osteocytes²⁴) was colocalized with cell lineage tracing to study chondrocyte transdifferentiation. If chondrocytes are revealed to be key players in condylar bone formation during late postnatal growth and directly respond to dietary loading, the focus of future studies should be shifted away from the bone marrow and instead be directed toward developing novel methods to control chondrocyte transdifferentiation. This could open new methods of research targeting condylar growth therapeutics and mandibular orthopedic therapies.

MATERIAL AND METHODS

The experimental protocol was approved by the Institutional Animal Care and Use Committee of Texas A&M School of Dentistry (IACUC 2022-0124-CD; reference no. 116605). Twelve 3-week-old male Aggrecan-Cre^ERT2 (Acan^Lineage); R26R^TdTomato; 2.3Col10a1-GFP and twelve 3-week-old male Col10a1-Cre (Col10a1^Lineage); R26R^TdTomato; 2.3Col10a1-GFP were randomly divided into 2 groups. Each group (n = 6) maintained normal mice chow (hard diet, the control group) or gel food (soft diet, the experimental group, ClearH20 76A, ME) for 6 weeks (Fig 1, A). Three-week-old mice are equivalent to prepubescent humans aged approximately 6-8 years.²⁵

Fig 1.

The experimental design in this study. A, Two types of compound mice: Aggrecan-Cre^ERT2; R26R^tdTomato; 2.3Col1a1-GFP and Col10a1-Cre; R26R^tdTomato; 2.3Col1a1-GFP were used. A soft diet was started at 3 weeks, and mice were harvested at 9 weeks. The tamoxifen injection was given to the Aggrecan-Cre^ERT2 line at 3 weeks; B, The bone quality of the condylar process superior to the inferior alveolar nerve was analyzed by μCT; C, The 2D measurements were analyzed with μCT images, and the following measurements were obtained: total mandibular length (Co-Me); mandibular base length (Me-Go); ramus height (Co-GoT); and condylar length (Co- Ic-Rd); D, The condylar process was divided into superior (light green) and inferior (light red) portions. The inferior portion was 250 μm thick, immediately inferior to the widest portion of the condyle as viewed in the frontal orientation. Co, condylion; Go, gonion; GoT, gonial tangent; Ic, the deepest point in the notch between condylar head and coronoid process; Me, menton; Rd, ramus depth.

One time tamoxifen (75 mg/kg) injections were given to Aggrecan-Cre^ERT2; R26R^TdTomato; 2.3Col1a1-GFP mice at 3 weeks. All mice were killed when aged 9 weeks with EdU (10 μg/mL, A10044; Invitrogen, Carlsbad, Calif) administered 3 hours before harvest. The sample size was based on published estimates assuming a power of 0.95 and α of 0.05, with a 10% difference and effect size of 2.4.²⁶

Mandibles were fixated in 4% paraformaldehyde, dissected, and separated in half along the midsymphysis. Microcomputed tomography (μCT) scans (μCT 35 scanner; Scanco Medical AG, Bassersdorf, Switzerland) were taken to quantify the bone quality of the condylar process beyond the mandible foramen (Fig 1, B).^13,27 The μCT 3-dimensional (3D) reconstruction of the hemimandibles was oriented consistently (to visualize all cusps of the first molar equally from the lingual view), and 2-dimensional (2D) images were captured of that orientation to measure total mandibular length, mandibular base length, ramus height, and condylar length with ImageJ (National Institutes of Health, Md) software by 1 examiner (A.J.) (Fig 1, C; Table I).^8,26,28

Table I.

Two-dimensional linear measurements

Measurements	Units	Soft diet	Hard diet	P value
Total mandibular length (Co-Me)	mm	9.53 ± 0.11	9.83 ± 0.16	0.004
Mandibular base length (Me-Go)	mm	8.65 ± 0.26	8.92 ± 0.21	0.006
Ramus height (Co-GoT)	mm	4.24 ± 0.15	4.36 ± 0.10	0.182
Condylar length (Co- ⊥lc-Rd)	mm	2.61 ± 0.07	2.63 ± 0.08	0.725

Note. Presented values are mean ± standard deviation.

The condylar process was decalcified in 10% ethylenediaminetetraacetic acid, dehydrated in 30% sucrose, and sectioned along the frontal plane parallel with the axis of the ramus. The slides from the middle third portion were used for the study. The sections for analysis were selected by 1 examiner in which the mediolateral width of the condyle was the largest. The sections were used for Toluidine blue, Safranin O staining,²⁹ or immunohistochemistry³⁰ (IHC) with the following antibodies: Aggrecan (the major proteoglycan in the articular cartilage,³¹ 1:400; Abcam, Cambridge, United Kingdom), Col10a1 (Collagen type 10a1 expressed by hypertrophic chondrocytes,³¹ 1:400; Abcam), and Sox9 (a key transcriptional factor for chondrocyte differentiation,^32,33 1:100; Abcam). Click-iT Imaging Kit with Alexa Flour 647-azide (A10277; Invitrogen) was used to detect EdU signal in cryosections for cell proliferation analysis.²⁹ Toluidine blue and Safranin O images were captured by a light microscope, and fluorescent images were captured using an SP5 Leica confocal microscope (Leica, Germany) at wavelengths ranging from 488 (green) to 561 (red) nm.³⁴

For chondrogenic activity, the EdU⁺ chondrocytes (EdU⁺ cells/area), the expression levels of Aggrecan, Col10a1 (marker⁺/area), and Sox9 in cartilage (Sox9⁺ cells/area) were quantified using ImageJ software.^35,36 Chondrocyte transdifferentiation was evaluated in the superior and inferior portions of the subchondral bone separately (Fig 1, D). The superior portion included the subchondral bone above the widest portion of the condyle, whereas the inferior portion was 250 μm thick, immediately below the widest portion of the condyle as viewed in the frontal orientation. The following parameters were quantified in the superior and inferior regions, as well as the combination of both areas: the numbers of Acan^Lineage or Col10a1^Lineage-derived bone cells (yellow and red), the number of nonchondrocyte-derived bone cells (green and blue), and the ratio of Acan^Lineage or Col10a1^Lineage-derived bone cells over all subchondral bone cells to represent the percentage of chondrocyte-derived osteogenesis.³⁰

Statistical analysis

All statistical analyses were conducted using SPSS (version 28.0; IBM, Armonk, NY). Based on the skewness and kurtosis statistics, the data were normally distributed. For quantitative analysis on the histologic level, 4 sections were used as an average for each animal. Parametric means and standard deviations were used to describe central tendency and dispersion. Between-group comparisons for condylar size were made using independent t tests. For 2D measurement, 3D μCT, IHC staining, and cell lineage tracing data, between-group comparisons were made using analysis of covariance, controlling for final mouse weight differences. The intraobserver error was assessed using intraclass correlation coefficients on repeated measures, with all analyses having an intraclass correlation >0.99.

RESULTS

There was no statistical significance between-group differences in weight before diet modification. After 1 week, the soft-food diet mice were significantly lighter (2-3 g) than the hard-food diet mice. This difference was maintained throughout the study (Table II).

Table II.

Weekly weights, along with probabilities of between-group differences

Week	Soft diet, g	Hard diet, g	P value
0	12.55 ± 1.12	11.88 ± 0.87	0.276
1	15.30 ± 2.00	17.70 ± 1.08	0.034
2	19.05 ± 1.22	21.89 ± 0.83	0.001
3	23.03 ± 1.58	25.24 ± 1.19	0.021
4	24.42 ± 2.23	27.74 ± 1.26	0.010
5	25.18 ± 2.31	28.68 ± 1.33	0.009
6	25.72 ± 2.36	29.55 ± 1.56	0.008

Note. Presented values are mean ± standard deviation.

The total mandibular length (Co-Me) and mandibular base length (Go-Me) were significantly shorter in the soft-food diet group than in the hard-food diet group (Table I). The difference in ramus height (Co-GoT) approached statistical significance, favoring the hard food group. The 3D μCT results showed no significant differences in the bone quality of the condylar process between groups, although the soft-diet condylar process displayed lower bone volume, total volume, and bone volume fraction, with the difference approaching statistical significance (Table III). In summary, the radiologic analysis revealed shorter mandibles with reduced bone quality among mice fed softer diets.

Table III.

μCT bone density, volume, and trabeculation of the condylar process

Measurements	Units	Soft diet	Hard diet	P value
Trabecular no.	1/mm	10.01 ± 0.70	9.44 ± 0.44	0.082
Trabecular thickness	mm	0.14 ± 0.01	0.14 ± 0.01	0.649
Trabecular spacing	mm	0.07 ± 0.00	0.06 ± 0.01	0.474
Total volume (TV)	mm3	1.09 ± 0.18	1.37 ± 0.21	0.178
Bone volume (BV)	mm3	0.78 ± 0.15	1.03 ± 0.15	0.113
BV fraction	NA	0.72 ± 0.03	0.75 ± 0.02	0.122
Mean/density of TV (apparent)	mg HA/ccm	707.76 ± 24.18	729.84 ± 24.73	0.332
Mean/density of BV (material)	mg HA/ccm	865.58 ± 18.05	871.31 ± 22.92	0.920

Note. Presented values are mean ± standard deviation.

NA, not applicable.

Both Safranin O (Fig 2, A) and Toluidine blue (Fig 2, B) images showed fewer layers of chondrocytes and decreased trabecular bone mass in the condylar process of the soft-diet group. The confocal images showed fewer EdU⁺ positive cells in the condyles of soft-diet mice than in hard diet-fed mice (Fig 2, C), and quantitative results confirmed that the number of EdU⁺ chondrocytes per unit condylar cartilage area was significantly lower in the soft-diet group (Fig 2, D). These data demonstrated that cell proliferation within the condylar cartilage was significantly lower in mice eating a softer diet.

Fig 2.

Histologic staining and cell proliferation for control and soft-diet groups. A and B, Safranin O (A) and Toluidine blue (B) images showed thinner condylar cartilage (double-head arrows) and less trabecular bone mass (arrows) in the soft-diet group; C, Confocal images for EdU staining displayed that the dividing cells (arrows) were dramatically decreased in soft-diet condyles; D, The quantitation data confirmed the significant decrease of EdU⁺ cells in the soft-diet group.

The confocal image of Sox9 IHC showed numerous Sox9⁺ cells evenly distributed in the control condylar cartilage, whereas much fewer Sox9⁺ cells were found in the soft-diet treatment (Fig 3, A, left). Quantifying the density of Sox9⁺ cells in the cartilage confirmed the significant reduction of Sox9 expression in the soft-diet group (Fig 3, A, right). Furthermore, there were significantly fewer yellow Acan^Lineage-differentiated Sox9⁺ chondrocytes in the soft-diet group (Fig 3, B). The level of Aggrecan expression was substantially lower in the condylar cartilage of the soft-diet group (Fig 3, C, left), with a significant reduction in the ratio of Aggrecan⁺ area to condylar cartilage area (Fig 3, C, right). Similarly, Col10a1 expression in the condyle was much lower with the soft than with the hard diet (Fig 3, D, left), with the ratio of Col10a1⁺ area to condylar cartilage area significantly decreased (Fig 3, D, right). In summary, these results showed that a consistent soft diet decreases chondrocyte proliferation, differentiation, and maturation.

Fig 3.

A soft diet leads to the reduction of condylar chondrogenic activity. A, The Sox9 IHC images and quantitation results showed significantly reduced Sox9⁺ cells (green) in the condyle cartilage of the soft-diet group; B, The Acan^Lineage-derived Sox9⁺ cells (yellow) were also significantly reduced in the soft-diet group; C, The Aggrecan IHC images showed a significant decrease (arrow) in the expression level of Aggrecan (green) in the soft-diet group, which was confirmed by the quantitation of Aggrecan positive area in the condyle cartilage; D, The Col10a1 IHC images showed a significant decrease in the expression level of Col10a1 (green) in soft-diet group (arrow), which was confirmed by quantitation of the Col10a1 positive area in the condyle cartilage.

Fluorescent confocal images of both Cre lines illustrated similar qualitative results (Figs 4, A and 5, A). In the subchondral bone of hard-diet condyles, there were numerous red and yellow chondrocyte-derived bone cells; the number of which was much lower in the soft-diet condyles. Furthermore, the number of yellow and red bone cells was greater in the superior region of the subchondral bone than in the inferior region in both food groups.

Fig 4.

Soft diet inhibits chondrocyte transdifferentiation in Acan^Lineage mice. A, The confocal images showed numerous Acan^Lineage-derived bone cells in yellow or red in both superior and inferior portions of control condyles, which were much decreased in the soft-diet condyles; B and C, The bar charts showed the quantitative results for the number of Acan^Lineage-derived, non-Acan^Lineage-derived bone cells (green or blue) and total bone cells, and the ratio of Acan^Lineage-derived bone cells to total bone cells in the condylar process (B), and within the superior and inferior regions (C), respectively. BC, bone cells.

Fig 5.

Soft diet inhibits chondrocyte transdifferentiation in Col10a1^Lineage mice. A, The confocal images showed numerous Col10a1^Lineage-derived bone cells in yellow or red in both the superior and inferior portions of control condyles, which noticeably decreased in the soft-diet group. B and C, The bar charts showed the quantitation for the number of Col10a1^Lineage-derived, non-Col10a1^Lineage-derived bone cells (green or blue) and total bone cells, and the ratio of Col10a1^Lineage-derived bone cells to total bone cells in the condylar process (B), and within the superior and inferior regions (C), respectively. BC, bone cells.

The quantitative results confirmed the qualitative results. Within the Aggrecan-Cre^ERT2; R26R^tdTomato; 2.3Col1a1-GFP genotype, the total number of Acan^Lineage-derived bone cells (yellow and red bone cells), as well as total bone cells in the superior and inferior regions, was significantly higher in the hard-diet group than in the soft-diet group, whereas the number of non-Acan^Lineage-derived bone cells was not significantly different between groups. This resulted in a insignificant difference in the ratio of Acan^Lineage-derived bone cells over total bone cells between groups (Fig 4, B). When separately analyzing cell transdifferentiation in the 2 regions, we found that Acan^Lineage-derived bone cells in the superior subchondral bone region were significantly less in the soft than in the hard-diet group. However, the number of nonchondrocyte-derived bone cells (green and blue bone cells) in the superior region was not significantly different between groups. The ratio of Acan^Lineage-derived bone cells to total bone cells in the superior region of the subchondral bone approached statistical significance, with the ratio being smaller in the soft-diet group. A similar trend was observed in the inferior region of the subchondral bone, although differences were less striking (Fig 4, C).

Col10a1^Lineage mice showed the same trend with Acan^Lineage in the numbers of chondrocyte-derived bone cells and the total bone cells and the ratio of chondrocyte-derived bone cells to total bone cells in the whole area of superior and inferior areas (Fig 5, A and B). When analyzing superior and inferior regions separately, the Col10a1^Lineage mice also showed similar trends with Acan^Lineage, except that the difference in the number of Col10a1^Lineage-derived bone cells in the inferior region was approaching a significant difference, and there was a significant reduction in the total number of bone cells in the inferior area of the soft-diet condylar process (Fig 5, C).

In summary, the cell lineage tracing results supported the notion that chondrocytes continuously transdifferentiate into bone cells and have a significant contribution to bone formation during late postnatal growth. Six-week soft diets greatly decreased chondrocyte transdifferentiation, whereas the number of nonchondrocyte-derived bone cells did not differ. As such, the total number of bone cells in the condylar process was significantly fewer.

DISCUSSION

A soft diet is a classic model to mimic decreased muscle loading to study its effects on craniofacial growth.^7,10,37 The present study found that a consistent decrease in dietary loading led to a reduced condylar and mandibular length (Table I), less bone volume and density (Table III), thinner condylar cartilage thickness, and less trabecular bone in the condylar process (Fig 2, A). These results are consistent with previous animal and human studies’ results. Furthermore, by using cell lineage tracing, this study, for the first time, demonstrated that chondrocyte-derived bone cells are the major source for the development of condylar bone during the adolescent stage, and reduced daily loading through a soft diet is directly responsible for a decrease in chondrocyte-derived osteogenesis within the mandibular condyle, resulting in shorter condylar process, smaller mandibles, and less bone volume and density.

It has been established that condylar chondrogenesis is altered in response to muscular loading.^27,38,39 This study showed significantly less chondrocyte cell proliferation (Fig 2, C and D) and reduced chondrocyte differentiation and maturation reflected by significantly reduced Sox9, Aggrecan, and Col10a1 expression within the condylar cartilage of soft-diet groups (Fig 3). In particular, the significant decrease in the density of Acan^Lineage-derived Sox9⁺ cells further confirmed that chondrocyte differentiation was impacted by the soft diet because the Acan⁺ Sox9⁺ chondrocytes were traced after tamoxifen injection at 3 weeks. These changes could be attributed to the adaptable nature of secondary cartilage and its response to altered loading conditions produced by diet modification. This study supported this aspect of secondary cartilage when muscle function is reduced with a softer diet.

The chondrogenic changes in response to loading have been well documented; however, the literature does not associate these changes with condylar bone changes, as chondrogenesis and osteogenesis were considered 2 separate processes. Through cell lineage tracing, this study demonstrated that chondrocyte transdifferentiation is active during late postnatal growth, which is directly regulated by dietary loading. Tracing results from the hard food group demonstrated that 66% and 76% of bone cells in the condylar process were derived from Acan^Lineage (Fig 4, B) or Col10a1^Lineage (Fig 5, B), respectively. More specifically, the proportions of bone cells that transdifferentiated from Acan^Lineage (Fig 4, C) or Col10a1^Lineage (Fig 5, C) were respectively 74% and 81% in the superior region and 51% and 67% in the inferior region. These results align with findings for early postnatal condylar development,¹⁵ indicating that chondrocyte-derived bone cells are still the major cell source for condyles during adolescent development.

The Acan^Lineage soft-diet mice had 30% fewer transdifferentiated bone cells in the superior region and 27% fewer in the inferior subchondral bone region (Fig 4, C), and in the Col10a1^Lineage soft-diet mice, there were 44% and 22% fewer transdifferentiated cells in the superior and inferior regions, respectively (Fig 5, C). Furthermore, there were fewer total bone cells in the condyles of mice fed soft diets from both Acan^Lineage (21% and 16% fewer bone cells in the superior and inferior portions, respectively; Fig 4, C) and Col10a1^Lineage (38% and 22% fewer bone cells in those respective portions; Fig 5, C). Significant differences between hard and soft-diet groups were found except in the inferior portion of Acan^Lineage mice. The differences in total bone cells between groups can be attributed to the contribution of transdifferentiated chondrocytes because the number of nonchondrocyte-derived bone cells was not significantly affected. This implies that the chondrocyte-derived osteogenesis pathway is responsible for fewer total bone cells during a consistent decrease in dietary loading. The percent differences in the number of transdifferentiated bone cells were very similar to the percent differences in chondrocyte cell proliferation (47% less; Fig 2, D) and maturation (30%-50% less; Fig 3), highlighting the continuous nature of this chondrocyte-to-bone-cell pathway.

The differences in the ratio of transdifferentiated chondrocytes to total bone cells within the condylar process approached significance in both Cre lines. In the condylar process, the percent of Acan^Lineage-derived bone cells to total bone cells in the hard and soft-diet group was 66% vs 57% (Fig 4, B); the percent of Col10a1^Lineage-derived bone cells to total bone cells in the hard and soft-diet group was 76% vs 71% (Fig 5, B). Within the superior region, the percent of Acan^Lineage- or Col10a1^Lineage-derived bone cells to total bone cells in the hard and soft-diet group was 74% vs 64% (Fig 4, C) or 81% vs 72% (Fig 5, C), respectively. Within the inferior region, these ratios were observed to be 51% vs 45% (Fig 4, C) or 67% vs 68% (Fig 5, C), respectively. The lack of significant differences in the ratios of transdifferentiated chondrocytes to total bone cells between groups in both Cre lines might be due to the significant decrease in the numbers of both chondrocyte-derived bone cells and the total bone cells. More studies with larger sample sizes are needed to confirm that the rate of chondrocyte transdifferentiation is statistically decreased with less dietary loading. These results implied that decreased dietary loading leads to reduced chondrocyte proliferation and maturation within the condylar cartilage, thus creating a smaller pool of chondrocyte cells to undergo transdifferentiation.

The cell lineage tracing results from Aggrecan-Cre^ERT2 and Col10a1-Cre lines showed very similar trends, which strongly confirmed the effects of dietary loading on the activity of chondrocyte transdifferentiation. The reason that more Col10a1^Lineage-derived bone cells were recorded is likely because of accumulated tracing from the embryonic stage, whereas the Acan^Lineage mice only traced those transdifferentiated bone cells after tamoxifen injection at 3 weeks.

The significant decrease in the number of transdifferentiated chondrocytes within the subchondral bone was likely to contribute to the observed differences in bone morphology and quality. This study demonstrated that a consistent soft diet produced significantly shorter mandibles with a 3.1% decrease in total length and a 3.0% decrease in base length. Although not statistically significant, the ramus height was 2.8% shorter in the soft-diet group (Table I), which is consistent with previous studies’ findings.^8,26,40 The minor difference in mandibular dimensions might be due to the age of the mice used in this study. Mouse mandible shape and size are expected to reach 95% maturity by age 35 days and 24 days, respectively.⁴¹ In addition, there were no significant differences in μCT measurements of the condylar process in this study, although multiple values approached significance, such as 24% less bone volume, 20% less total volume, and 4% smaller bone volume fraction in soft-diet group (Table III). These percent differences were similar to those in previous studies,^8,9,42,43 indicating a trend of decreases in bone quality and volume.

It has been suggested that increasing bone mass is an adaptation to better withstand higher functional demands, and a larger condyle in response to higher loads supports this hypothesis^44,45 The 2D and 3D mCT and histology results in this study demonstrated more bone formation in the condylar process among mice fed a hard diet. Through the use of cell lineage tracing, this study was able to attribute those differences to chondrocyte-derived osteogenesis. Therefore, we can assume that chondrocyte-derived bone formation is the pathway responsible for adaptations to functional changes.

In summary, this is the first study to explain why dietary loading during adolescent growth is so important for mandibular morphology and how it affects mandibular condyle growth. Our results demonstrated that the physical consistency of the diet regulated not only condylar chondrocyte proliferation and maturation, as previously reported, but also the cell transdifferentiation from chondrocytes to bone cells (Fig 6). Because the chondrocyte-derived bone cells are the major cell source of condylar bone in the adolescent stage, it could be a promising strategy to control the condyle growth under the regulation of dietary loading on chondrocyte transdifferentiation in the future orthodontic and orthopedic treatments. Currently, the effect of changes in masticatory loading on the mechanical stimulation of the condyle is still unclear. Further studies are necessary to investigate how forces applied to the surface of the condyle could impact chondrocyte transdifferentiation.

Fig 6.

The diagram for working hypothesis: chondrocytes play a key role in the endochondrogenesis of the temporomandibular joint condyle in adult-aged mice by transdifferentiating into bone cells, and physical consistency of the diet directly affects chondrogenesis and chondrocyte-derived osteogenesis, resulting in normal mandibular growth.

Patients with hyperdivergence and retrognathia are among the most challenging phenotypes to treat orthodontically. These patients have functional concerns related to weak musculature and poor masticatory performances, which will not self-correct and could worsen over time.^46–52 Clinical studies have shown that adolescent patients undergoing masticatory muscle training experienced greater forward mandibular rotation and an improvement in their skeletal open bites.^6,53 This study suggested that targeting the chondrocyte-derived osteogenesis pathway through dietary loading opened up therapeutic possibilities, and orthodontists may have the ability to manipulate condylar growth through muscle strengthening.

Throughout a patient’s orthodontic treatment, the entire orofacial system is put through a multitude of changes, leading to a softer diet and less masticatory activity with a significant reduction in the size of the masseter muscle.⁵⁴ In adult patients or hypodivergent adolescent patients, this is likely not a concern. However, in hyperdivergent retrognathic patients, it may be detrimental to lower masticatory muscle activity throughout treatment, and it may be beneficial to offset the inevitable change in masticatory muscle function with strengthening exercises or hard gum chewing. The present study helped illustrate why daily masticatory activity plays a role in mandibular and condylar development. Chondrocytes are the major cell contributors to the condylar bone during adolescent growth and are shown to be regulated by dietary loading. Therefore, emphasis should be placed on maintaining masticatory muscle activity during adolescent orthodontic treatment.

CONCLUSIONS

A 6-week decrease in dietary loading on mice results in the following: (1) less chondrocyte proliferation within the condylar cartilage, (2) less chondrocyte maturation within the condylar cartilage, (3) fewer chondrocyte-derived bone cells and total bone cells within the subchondral bone of the condyle, and (4) shorter mandibular length.