Stage-Specific Expression of Histone Deacetylases in the Mouse Mandibular Condylar Cartilage

Abstract

Objective

This study investigates the relationship between aging and histone deacetylases (HDACs) in mandibular condylar cartilage.

Methods

Male C57BL/6 mice were divided into 4 age groups: postnatal day 21 (D21, postnatal development), 3 months (3M, young adulthood), 10 months (10M, middle aging), and 18 months (18M, late aging) to cover mandibular condylar cartilage aging stages. Mandibular condylar cartilage specimens were harvested and paraffin-embedded. Hematoxylin-eosin (H&E) and Safranin O-fast green staining were performed to assess morphological changes. Immunohistochemistry (IHC) was applied to map the spatiotemporal expression of the HDAC1-11 in condylar cartilage.

Results

HDAC1, HDAC6, HDAC7, and HDAC9 were undetected in the mandibular condylar cartilage. HDAC2, 3, 4, 8, and 10 were only expressed 21 days after birth, with HDAC2 showing the strongest expression. Notably, HDAC5 expression was higher at 18 months. HDAC11 had consistent intensity until 18 months when it decreased. Other HDACs peaked at 21 days and declined with age.

Conclusion

This study established the spatiotemporal expression pattern of HDAC1-11 in the temporomandibular joint (TMJ) condyle during aging. It provides a foundation for further research on HDAC functions, offering insights into TMJ aging mechanisms.

Keywords

histone deacetylases expression pattern temporomandibular joint mandibular condylar cartilage

Introduction

The temporomandibular joint (TMJ), a remarkable and intricate anatomical structure, serves as the linchpin for the complex biomechanics of mastication.¹ At the heart of this joint lies the condyle, a structure of paramount importance. Composed of a unique blend of cartilage and underlying bone, the TMJ condyle is tasked with withstanding the repetitive forces and stresses generated during the act of chewing, speaking, and a myriad of other oro-facial movements.^1
-3 It is not merely a passive component; rather, it is a dynamic entity that adapts and responds to the ever-changing demands placed upon it throughout an individual’s life.

As the body ages physiologically, the TMJ condyle faces multiple challenges.⁴ Age-triggered changes within the condyle run deep, affecting both microstructure and function.^5,6 The once-flexible cartilage now exhibits signs of deterioration,⁷ with alterations in its matrix and cell viability. Meanwhile, the underlying bone undergoes remodeling, which may be maladaptive, weakening the condyle’s overall integrity. These changes have a domino effect, potentially impeding masticatory efficiency and leading to pain, reduced jaw mobility, and even facial deformities in the elderly.⁸

Epigenetic changes are regarded as one of the hallmarks of aging, which has been confirmed by many studies.^9
-11 Epigenetic modifications do not alter DNA sequence, instead, they alter DNA accessibility and chromatin structure, which in turn regulate gene expression patterns.¹⁰ In the quest to understand the molecular underpinnings of TMJ disorder (osteoarthritis) in the TMJ condyle, epigenetics has now become a focus of attention. Ukita et al.¹² demonstrated that histone H3K9 methylation regulates chondrocyte homeostasis in terms of cell growth, apoptosis, and gene expression. Liao et al.^13,14 detected the effect of histone deacetylases (HDACs) on TMJ synovium-derived mesenchymal stem cell inflammatory activation. However, the research on the relationship between the subtypes of HDACs and mandibular joint is still relatively few.

HDACs—an enzyme family long recognized for regulating gene expression—hold the key to chromatin remodeling and transcriptional control, and consist of multiple subtypes with distinct structural and functional characteristics ( Table 1 ).¹⁵ Alterations in the expression levels of several HDACs induce alterations in histone acetylation, which are implicated in the regulation of lifespan in different organisms.¹⁶ Class I HDACs, which include the likes of HDAC1, HDAC2, and HDAC3, have emerged as guardians of genomic stability in various tissues. Patnaik et al.¹⁷ suggested that HDAC1 might be a novel therapeutic target aiming to protect genomic integrity in the context of neurodegeneration and aging. Class II HDACs, further divided into Class IIa and Class IIb, present their own set of complexities.^18,19 Class IIa HDACs, such as HDAC4, HDAC5, and HDAC7, are known for their ability to shuttle between the nucleus and cytoplasm, thereby modulating gene expression in a context-dependent manner.¹⁵ In the realm of cardiac aging, alterations in their subcellular localization and activity had profound implications for cardiomyocyte function.^20,21 However, a comprehensive grasp of the expression patterns of HDACs throughout the physiological aging process of the TMJ, particularly within the cartilage, remains unclear. Therefore, in this study, we aimed to investigate the relationship between aging and HDAC1-11 in mandibular condylar cartilage.

Table 1.

HDACs in Mice Skeletal Development and Bone Metabolism: KO/CKO Phenotypes and Functions.

Classification	HDAC	KO/CKO	Function	Reference (PMID)
Class I	HDAC1	KO	Embryonic lethal	12032080
	HDAC2	KO	Reduced body size, smaller vertebrae, and pelvis	17909008
	HDAC3	KO	Embryonic lethal (E9.5)	18830415
		Osx1-Cre	Cortical and trabecular bone loss	23408427
			Decreased bone formation rate
			Fewer osteoblasts, increased marrow adiposity
			Abnormal chondrocyte hypertrophy
		OCN-Cre	Age-related cortical and trabecular bone loss	23085085
			Decreased bone formation rate
			Reduced osteoblast numbers
		Wnt1-Cre	Craniofacial abnormalities	23506836
		Pax3-Cre	Craniofacial abnormalities	23506836
		Col2-Cre	Embryonic lethal	27507649
Class II	HDAC4	KO	Ectopic chondrocyte hypertrophy	20097749
			Enhanced endochondral ossification
			Increased MMP13 expression
		Runx2-Cre	Reduced bone density	25685691
			Increased Rankl expression and bone resorption
			Reduced osteocalcin expression
	HDAC5	KO	No gross skeletal phenotypes at birth	15367668
	HDAC6	KO	Increased trabecular bone mineral density	18180281
	HDAC7	KO	Embryonic lethality	16873063
		Tie2-Cre	Embryonic lethality	23204328
		Col2a1-Cre	Embryonic lethality	25389289
			(Reduced trabecular bone volume and trabecular number
			increased chondrocyte proliferation)
		LysM-Cre	Reduced bone mass	23204328
			Increased bone resorption
			Normal bone formation
		PT-Cre	Reduced bone mass	23204328
			Increased bone resorption
			Normal bone formation
Class I	HDAC8	KO	Ossification defects in frontal and interparietal bones	19605684
Class II	HDAC9	KO	Reduced bone mass	25793404
			Increased bone resorption
			Reduced bone formation
Class IV	HDAC11	KO	No gross skeletal phenotypes	25155994

Materials and Methods

Animals

C57BL/6J genetic background male mice were obtained from Huachuang Xinnuo (Jiangsu, China). All animal procedures strictly followed the 3Rs (Replacement, Reduction, Refinement) principles. Mice were housed in a facility in a 12-h light-dark cycle with controlled temperature (21°C ± 1.5°C) and humidity (50% ± 10%) and had access to food and water ad libitum. Mouse tissues were collected from ages 21 days, 3 months, 10 months, and 18 months. At each time point, mice (n = 3 per group) were euthanized by intraperitoneal injection of sodium pentobarbital (100 mg/kg body weight) following the American Veterinary Medical Association (AVMA) guidelines. Immediately after euthanasia, the entire mandible (including the mandibular condyle) was dissected from each mouse using sterile surgical scissors and forceps. The surrounding soft tissues (e.g., muscle, fascia, and skin) were carefully removed under a stereomicroscope to avoid contamination of the condylar tissue. The dissected mandibles were rinsed with cold phosphate-buffered saline (PBS, pH 7.4) 3 times to remove blood and debris before fixation. Immediately after tissue collection, a supplementary dose of sodium pentobarbital (100 mg/kg body weight, i.p.) was administered to the anesthetized mouse to ensure euthanasia. The mouse was then placed in a quiet, warm environment and continuously monitored for 5 min to confirm the completion of euthanasia: absence of respiratory movement, loss of corneal reflex, and no cardiac pulse. Only after all these signs were confirmed was the euthanasia process considered complete. This study was reviewed and formally approved by the Animal Ethics Committee of the Second Affiliated Hospital of Harbin Medical University, with the official approval number: YJSDW2025-241.

Histological Staining

The mouse mandible was immersed in 4% paraformaldehyde (PFA) fixative (Solarbio, Beijing, China) at 4°C for 24 h to achieve complete protein coagulation. After fixation, the mandible was transferred to a 10% ethylenediaminetetraacetic acid (EDTA, pH 7.4) decalcifying solution, which was refreshed every 3 days. Decalcification progress was evaluated every 7 days using the needle puncture test (a standard histopathological method for soft tissue-bone complex decalcification confirmation): A sterile 21G hypodermic needle was gently inserted into the mandibular condyle (the region with the thickest subchondral bone) with uniform, moderate force. If the needle penetrated the tissue smoothly without resistance (i.e., no “hard rebound” feeling from undecalcified bone) and the tissue surface showed no obvious needle-induced cracking, decalcification was considered complete. At the end of decalcification, the mandible was dehydrated through a graded ethanol series (70%, 80%, 90%, 95%, and 100% ethanol, 1 h each) and cleared in xylene (2 × 1 h). The mandible was then embedded in melted paraffin (56°C-58°C) in a paraffin-embedding machine (Thermo Fisher Scientific), with the mandibular condyle oriented coronally to ensure that sections contained the full thickness of the condylar cartilage and underlying subchondral bone. Three-micrometer-thick sections were prepared using a microtome (Thermo Fisher Scientific) and mounted on poly-l-lysine-coated glass slides—this coating was used to prevent section detachment during subsequent staining procedures. The sections were then stained separately with hematoxylin and eosin (H&E) and Safranin O-Fast Green (staining kits from Solarbio) following the manufacturer’s standard protocols. Thickness measurements were performed using Image J (National Institutes of Health). The software was first calibrated with the slide’s built-in scale bar (50 μm) for pixel-to-μm conversion. For the proliferative zone (PZ), thickness was defined as the perpendicular distance between 2 clear boundaries: the lower edge of the fibrous zone (FZ, where flattened cells transition to spindle-shaped cells) and the upper edge of the mature zone (MZ, where spindle-shaped cells transition to rounded cells). Three independent measurements were taken for the PZ layer in the middle region of each section, and the average value was used for statistical analysis. All measurements were conducted by 2 researchers blinded to animal age, with an inter-observer correlation coefficient (ICC) > 0.9 confirming measurement consistency.

Immunohistochemistry

IHC staining was performed on 3-μm-thick whole tissue sections. Sections were deparaffinized to water, antigen repaired (sodium citrate heat-induced, about 120°C), H₂O₂ blocked and 3% bovine serum albumin (BSA) blocked. IHC was performed with the use of the following antibodies: anti-HDAC1-HDAC11 ( Table S1 ). Slides were incubated with antibodies overnight at 4° C. The next day, the tissues were washed 3 times in PBS for 3 min each time. The secondary antibody was applied for about 30 min at room temperature. Immunostaining was performed by incubating with 3′-3-diaminobenzidine (DAB) for 5 min, followed by a 1 min counterstain with hematoxylin. Each tissue slide contained one tissue as a negative control on the slide. IHC staining for all age groups (D21, 3M, 10M, 18M) was performed in a single batch. This included identical reagents (primary/secondary antibodies, DAB, blocking solutions), incubation times/temperatures, and equipment (same microwave, humidified incubator) to eliminate batch effects. For semi-quantitative analysis, 3 non-overlapping representative regions were selected from the condylar cartilage and subchondral bone of each sample. The average optical density (AOD) of the DAB-stained positive areas was measured using Image J software²² (National Institutes of Health). The AOD value was calculated as the ratio of the integrated optical density (IOD) of positive signals to the area of the selected region, which reflects the relative expression level of HDACs. For each HDAC subtype, measurements were performed on 3 independent tissue sections per age group (n = 3 mice per group) to ensure statistical reliability.

Statistical Analysis

All continuous variables were tested for normal distribution by the Shapiro-Wilk test, and the data consistent with normal distribution were analyzed using 1‑way analysis of variance followed by Tukey’s multiple comparison test to determine statistical differences between groups. P < 0.05 was considered to indicate a statistically significant difference.

Results

Histological Change

To elucidate age-related structural changes in the mouse mandibular condyle, we performed H&E and Safranin O-Fast Green staining at 4 key time points: D21, 3M, 10M, and 18M (Figs. 1 and 2).

Figure 1.

H&E staining of condyle and condylar cartilage. At 10 months, the proliferative zone became thinner than 3 months and from postnatal day 21 (D21) (E, F, and G). However, at 18 months, the proliferative zone was not very clear (H). In addition, at 18 months, the hypertrophic zone was thin and even disappeared. D21 (A), 3 months (B), 10 months (C), and 18 months (D) condylar process; D21 (E), 3 months (F), 10 months (G), and 18 months (H) condylar cartilage; D21 (I), 3 months (J), 10 months (K), and 18 months (L) subchondral bone. (A), (B), (C), and (D) Low-magnification view (10×, scale bar = 100 μm); (E), (F), (G), and (H): high-magnification view of condylar cartilage (40×, scale bar = 50 μm); (I), (J), (K), and (L): high-magnification view of the subchondral bone (40×, scale bar = 50 μm).

Figure 2.

Safranin O-Fast Green staining of condyle and condylar cartilage. D21 to 18 months (18M) (A-D), condylar cartilage and underlying bone tissue showed dynamic changes, transitioning from “development and maturation” to “structural stability and remodeling,” with details of the cartilage layer (E-H) and bone tissue (I-L) as follows. In the cartilage layer: D21 (E) showed an intact 4-layer structure (fibrous zone[FZ], proliferative zone [PZ], mature zone [MZ], hypertrophic zone[HZ]) with dense PZ cells and regularly arranged HZ cells; 3M/10M (F-G) exhibited gradual thinning, obvious HZ degeneration (reduced, loosely arranged cells), and decreased PZ cell density (weakened proliferation); 18M (H) showed further thinning, near-disappearance of HZ in some areas, blurred layer boundaries, and denser PZ fibrous layer. In the underlying bone tissue: D21 (I) had thin, sparse trabeculae (reticular distribution), large loose marrow cavities, and abundant osteoblasts on trabecular surfaces; 3M/10M (J-K) showed thickened, denser trabeculae with increased branching and regular networks; 18M (L) presented mature trabeculae with uniform thickness and stable density. OCY: Osteocyte; BM: Bone marrow; TB: Trabecular bone; OB: Osteoblast; OC: Osteoclast. (A), (B), (C), and (D): Low-magnification view (10×, scale bar = 100 μm); (E), (F), (G), and (H): high-magnification view of condylar cartilage (40×, scale bar = 50 μm); (I), (J), (K), and (L): high-magnification view of the subchondral bone (40×, scale bar = 50 μm).

At D21, the mandibular condyle exhibited a well-organized architecture across its 3 compartments (condylar process, condylar cartilage, and subchondral bone). The condylar cartilage was clearly divided into 4 distinct zones (fibrous, proliferative, mature, and hypertrophic) with dense, morphologically distinct chondrocytes in each zone. The subchondral bone displayed a robust trabecular network populated by abundant osteocytes and active bone-forming cells. As aging progressed, progressive structural deterioration became evident. In the condylar process, tissue organization gradually disrupted; within the condylar cartilage, chondrocyte density decreased significantly, particularly in the FZ and PZs (Fig. 1).

Safranin O-Fast Green staining further revealed age-dependent alterations in extracellular matrix composition and cellular distribution. At D21, the mature and hypertrophic zones (HZs) of the condylar cartilage showed intense Safranin O staining, indicative of high proteoglycan content. While the zone boundaries remained discernible at 3M, proteoglycan staining in the HZ exhibited a mild reduction. By 10M, the proliferative and HZs were notably attenuated, and proteoglycan content in the MZ decreased substantially. At 18M, the PZ was barely identifiable, the HZ was largely absent, and proteoglycan staining was confined to scattered areas in the MZ. In the subchondral bone, osteoid matrix and osteocyte density showed progressive decline: at D21, trabecular bone was dense with active osteoblasts and osteocytes; at 3M, osteoclasts appeared, indicating ongoing bone remodeling; at 10M and 18M, trabecular bone thickness diminished further, and osteocyte density was significantly reduced, with 18M samples exhibiting severe bone matrix degradation (Fig. 2).

Quantitative assessment of the PZ thickness confirmed these qualitative observations. The PZ thickness showed a dynamic trend with aging: it remained stable from D21 to 3M, followed by a significant reduction at 10M compared with the younger groups, and this thinning became more pronounced by 18M. Statistical analysis indicated extremely significant differences between all age groups (P < 0.0001, Figs. S1), underscoring the pronounced age-dependent attenuation of the PZ. In addition, the HZ was severely attenuated in 18-month-old mice, with occasional regional absence. Collectively, these findings demonstrate that the mouse mandibular condyle undergoes progressive structural degeneration with aging, involving both cartilaginous and osseous compartments.

Immunohistochemistry

To elucidate the role of HDACs in mandibular condylar cartilage, IHC was performed to examine the spatial localization of various HDAC subtypes in the mandibular condylar cartilage of mice at different postnatal stages, spanning from D21 to 18M. The distinct expression patterns of these HDAC subtypes are summarized in Figures 3 and 4 and Tables S2 and S3 .

Figure 3.

Schematic diagrams of anatomical localization and histological zoning of mandibular condylar cartilage, and heatmap of HDAC subtype expression across different age groups. (A) and (B) Integrated schematic diagram showing the anatomical location of the mandibular condylar process in the temporomandibular joint and age-related histological zoning of the condylar cartilage (right panel, postnatal day 21 [D21], 3 months [3M], 10 months [10M], 18 months [18M]). Fibrous zone (FZ), proliferative zone (PZ), mature chondrocyte zone (MZ), hypertrophic zone (HZ), indicating age-related changes: gradual thinning of PZ (3M vs. D21) and extreme attenuation of PZ (18M vs. 10M), consistent with histological staining observations. (C) Heatmap showing the relative expression levels of HDAC subtypes (HDAC2, HDAC3, HDAC4, HDAC5, HDAC7, HDAC8, HDAC10, HDAC11) in the mandibular condylar cartilage of mice at D21, 3M, 10M, and 18M. The color intensity represents the normalized expression value (range: −1 to 0.5), with darker colors indicating higher expression.

Figure 4.

Semi-quantitative analysis of HDAC subtype expression in the mandibular condylar cartilage and subchondral bone of mice across different age groups. (A)-(G) Quantitative analysis of HDAC2, HDAC3, HDAC4, HDAC5, HDAC8, HDAC10, and HDAC11 expression in the mandibular condylar cartilage. (H)-(N) Quantitative analysis of HDAC2, HDAC3, HDAC4, HDAC5, HDAC8, HDAC10, and HDAC11 expression in the subchondral bone. In all panels: The x-axis represents the 4 age groups (D21, 3M, 10M, 18M); the y-axis represents the relative expression level (AOD) value. Statistical significance was analyzed by 1-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Mandibular condylar cartilage

Surprisingly, our findings revealed distinct expression patterns of HDACs in the mandibular condylar cartilage and subchondral bone across different age groups of mice. First, HDAC1, HDAC6, HDAC7, and HDAC9 were not detectable in both the condylar cartilage and subchondral bone throughout the entire study period ( Figs. S2-S5 ). Among the remaining HDAC subtypes, those belonging to Class I and Class II were only expressed in the young mouse group at D21. As the mice aged from 3M to 18M, the expression of these HDACs was no longer detectable.

Focusing on D21 mice, HDAC2 (Class I HDACs) was predominantly localized in the nuclei of chondrocytes in the MZ and HZ of the condylar cartilage ( Fig. 5 ). Notably, HDAC2 expression peaked at D21—a time point when the condylar cartilage exhibited robust chondrocyte proliferation in the PZ and dense proteoglycan deposition in the MZ (Figs. 1E and 2E)—and was undetectable in mice aged 3M to 18M ( Fig. 4A ). Their reduced expression in aged cartilage coincided with the thinning of the HZ and decreased chondrocyte hypertrophy. Meanwhile, HDAC3, HDAC8, and HDAC10 exhibited moderate expression levels (Figs. 6 -8). Notably, HDAC10 was not detected in the surface zone ( Fig. 8 ), whereas HDAC3 and HDAC8 were present in the surface zone of the condyle, the fibrochondral zone, and the subchondral bone region ( Figs. 6 and 8 ).

Figure 5.

Immunohistochemical expression of HDAC2 in mouse mandibular condylar cartilage across different age groups.

Figure 6.

Immunohistochemical expression of HDAC3 in mouse mandibular condylar cartilage across different age groups. Immunohistochemical staining images of HDAC3 in the mandibular condylar cartilage of mice at postnatal day 21 (D21, A, E, and I), 3 months (3M, B, F, and J), 10 months (10M, C, G, and K), and 18 months (18M, D, H, and L). HDAC3 is predominantly expressed in the cytoplasm of PZ cells, whereas in the Mature Chondrocytes Zone (MZ), its expression is primarily localized to the cell nucleus. In addition, HDAC3 is mainly expressed in the subchondral bone at the D21 stage. (A), (B), (C), and (D): Low-magnification view (10×, scale bar = 100 μm); (E), (F), (G), and (H): high-magnification view of condylar cartilage (40×, scale bar = 50 μm); (I), (J), (K), and (L): high-magnification view of the subchondral bone (40×, scale bar = 50 μm).

Figure 7.

Immunohistochemical expression of HDAC8 in mouse mandibular condylar cartilage across different age groups. Immunohistochemical staining images of HDAC8 in the mandibular condylar cartilage of mice at postnatal day 21 (D21, A, E, and I), 3 months (3M, B, F, and J), 10 months (10M, C, G, and K), and 18 months (18M, D, H, and L). HDAC8 is mainly expressed in the FZ region of the cartilage at D21, the cytoplasm of the PZ region, and the nucleus of the MZ region. (A), (B), (C), and (D): Low-magnification view (10×, scale bar = 100 μm); (E), (F), (G), and (H): high-magnification view of condylar cartilage (40×, scale bar = 50 μm); (I), (J), (K), and (L): high-magnification view of the subchondral bone (40×, scale bar = 50 μm).

Figure 8.

Immunohistochemical expression of HDAC10 in mouse mandibular condylar cartilage across different age groups. Immunohistochemical staining images of HDAC10 in the mandibular condylar cartilage of mice at postnatal day 21 (D21, A, E, and I), 3 months (3M, B, F, and J), 10 months (10M, C, G, and K), and 18 months (18M, D, H, and L). HDAC10 is expressed in the cytoplasm of cells in the PZ region of cartilage, in the nucleus of cells in the mature zone (MZ), and in osteoblasts in the subchondral bone. (A), (B), (C), and (D): Low-magnification view (10×, scale bar = 100 μm); E, F, G, and H: high-magnification view of condylar cartilage (40×, scale bar = 50 μm); I, J, K, and L: high-magnification view of the subchondral bone (40×, scale bar = 50 μm).

Regarding Class II HDACs: HDAC4 showed weak expression specifically in the nuclei of condylar chondrocytes (Fig. S6). In contrast, HDAC5 exhibited moderate expression in the HZ of condylar chondrocytes, and this expression was specifically observed in 18-month-old (18M) mice ( Fig. 9 ). This age-specific pattern coincided with severe attenuation of the HZ and reduced subchondral bone trabecular density in 18M mice (Figs. 1H and 2L).

Figure 9.

Immunohistochemical expression of HDAC5 in mouse mandibular condylar cartilage across different age groups. Immunohistochemical staining images of HDAC5 in the mandibular condylar cartilage of mice at postnatal day 21 (D21, A, E, and I), 3 months (3M, B, F, and J), 10 months (10M, C, G, and K), and 18 months (18M, D, H, and L). HDAC5 exhibited moderate expression in the cytoplasm of chondrocytes within the hypertrophic zone, and this expression pattern was exclusively observed in 18-month-old mice. In addition, HDAC5 is predominantly expressed in the subchondral bone and bone marrow, with the highest expression level detected at 18 months. (A), (B), (C), and (D): Low-magnification view (10×, scale bar = 100 μm); (E), (F), (G), and (H): high-magnification view of condylar cartilage (40×, scale bar = 50 μm); (I), (J), (K), and (L): high-magnification view of the subchondral bone (40×, scale bar = 50 μm).

HDAC11 was highly expressed in the condylar cartilage from D21 up to 10 months of age; however, its expression declined markedly at 18 months. In addition, in the subchondral region, HDAC11 consistently displayed weaker expression compared with other HDAC subtypes ( Fig. 10 ). These detailed expression profiles provide crucial insights into the potential roles of HDACs in the development and aging of the mandibular condylar region.

Figure 10.

Immunohistochemical expression of HDAC11 in mouse mandibular condylar cartilage across different age groups. Immunohistochemical staining images of HDAC11 in the mandibular condylar cartilage of mice at postnatal day 21 (D21, A, E, and I), 3 months (3M, B, F, and J), 10 months (10M, C, G, and K), and 18 months (18M, D, H, and L). At 21 days, HDAC11 is expressed in the cytoplasm of fiber zone (FZ) cells, the nucleus of proliferative zone (PZ) cells, and partially in the cytoplasm of hypertrophic zone (HZ) cells. In the bone marrow and bone cells beneath the cartilage. In the cytoplasm of the PZ zone of cartilage, the nucleus of the mature zone (MZ), and the osteoblasts in the bone beneath the cartilage. At 3 and 10 months, HDAC11 is expressed in the cytoplasm of the PZ zone, the nucleus of the MZ and HZ zones, and in the osteoblasts in the bone beneath the cartilage. At 18 months, the expression of HDAC11 weakens, mainly expressed in the nucleus of the MZ cells, and weakly expressed in the cytoplasm of the bone cells beneath the cartilage. (A), (B), (C), and (D): Low-magnification view (10×, scale bar = 100 μm); (E), (F), (G), and (H): high-magnification view of condylar cartilage (40×, scale bar = 50 μm); (I), (J), (K), and (L): high-magnification view of the subchondral bone (40×, scale bar = 50 μm).

Discussion

In recent years, HDACs have increasingly captured attention due to their significant roles in diverse organs and tissues, including cartilage. In this study, we establish the first comprehensive spatiotemporal expression profile of HDAC1-11 in aging mouse mandibular condylar cartilage: (1) HDAC1, 6, 7, 9 were undetectable across all age groups (D21, 3M, 10M, 18M); (2) HDAC2, 3, 4, 8, 10 were expressed exclusively at D21 (with HDAC2 showing the strongest signal); (3) HDAC5 was expressed only at 18M; (4) HDAC11 was highly expressed from D21 to 10M but declined at 18M. These expression patterns correlate with age-dependent structural deterioration of the condylar cartilage (e.g., PZ thinning, reduced proteoglycan content) and subchondral bone remodeling. Moreover, the number of chondrocytes and the thickness of cartilage gradually declined with the aging of mice. However, there was minimal difference in cartilage thickness between 10- and 18-month-old mice. These age-related alterations are analogous to those of human condyle. Since late puberty, the superficial layer of cartilage has begun to exhibit age-related changes.²³ The superficial zone of cartilage is the most vulnerable to aging.^24
-26

Class I HDACs (HDAC1, 2, 3, and 8) have a tendency to inhibit chondrogenesis by suppressing chondrocyte-specific genes. The knockdown of HDAC1 and HDAC2 led to an increase in cartilage gene expression.²⁷ Despite their high degree of homology,²⁸ HDAC1 and HDAC2 display completely different expression patterns in the condyles of 3-week-old mice. In the present study, HDAC1 showed no expression at all, while HDAC2 exhibited the strongest expression among HDAC1-11. This suggests that HDAC1 and HDAC2 can target different genes to regulate chondrocytes. Previous reports showed that HDAC2 and HDAC8 were highly expressed in the HZ of the forelimb at embryonic day 16.5, whereas the expression of HDAC3 was weak.²⁹ However, at 21 days after birth, the expression levels of HDAC3 and HDAC8 in the MZ were nearly the same, and the expression of HDAC2 was extremely strong. Perhaps this difference in expression is related to both time and location. Bradley et al.³⁰ showed that the expression of HDAC3 is higher in prehypertrophic chondrocytes and lower in peripheral proliferating and hypertrophic chondrocytes at 4 weeks after birth. This is in line with our findings in the condyle. It has also been demonstrated that the depletion of HDAC3 can delay or prevent the terminal differentiation of chondrocytes in HDAC3-CKO_Osx mice.³¹ Studies have utilized postnatal conditional knockdown of HDAC7 to explore the role of HDAC7 in endochondral osteogenesis. They found that the deficiency of HDAC7 increased the number of proliferating chondrocytes and decreased the number of cells in hypertrophic areas.³² Another study has demonstrated that HDAC3 can promote osteoclast differentiation, while HDAC7 can inhibit osteoclast differentiation.³³ We found that the expression of HDAC3 at 21 days was higher than that of HDAC7 in the maturation zone of the condyle. Therefore, the specific mechanism of HDAC3 and HDAC7 in condylar cartilage is worthy of further study.

In our study, we discovered that HDAC4 was localized in the nuclei of hypertrophic cartilage within the condylar cartilage. It has been reported that HDAC4 is expressed in prehypertrophic chondrocytes.²⁷ In HDAC4 mutant mice, chondrocyte hypertrophy accelerates, resulting in premature endochondral mineralization.³⁴ We found that the expression of HDAC4, HDAC5, and HDAC10 in the mouse condyle was concentrated in the mature and hypertrophic areas at 21 days after birth. Surprisingly, HDAC6 and HDAC9 were not even expressed at all. Reportedly, the phenotype of rib chondrocytes, tibial growth plates, and the entire tibia were normal in HDAC5-deficient mice.³⁵ Previous studies also failed to identify skeletal abnormalities in mice lacking HDAC9.^36,37 However, when HDAC5 and HDAC9 are mutated together, the growth of mice slows down.³⁷ These results and our research on the condyle might suggest that the regulatory roles of HDAC5 and HDAC9 in the condyle are weak. However, the bone phenotype of HDAC4^-/- mice is highly similar to the cardiac phenotype of HDAC9^-/-mice,³⁴ indicating that class II HDACs may have comparable regulatory mechanisms in different cell types. Furthermore, the expression of HDAC5 in the mouse condyle was higher at 18M than at D21. This implies that HDAC5 may be associated with the regulation of condylar aging. HDAC5 functions as a transcriptional repressor that negatively regulates osteoclast differentiation by suppressing Nfatc1, Dc-stamp, and Ctsk, which are essential for osteoclast fusion and resorptive activity.^38
-41 Therefore, the increased HDAC5 expression observed at 18M may reflect an adaptive response to aging-related mechanical and metabolic stress, aiming to restrain excessive bone resorption and maintain cartilage-bone homeostasis. In contrast, Hdac11 knockdown selectively elevates Dc-stamp expression⁴² and is linked to deacetylation of the IL-10 promoter,⁴³ indicating a role in fusion and inflammatory regulation. The decline of HDAC11 may indicate weakened epigenetic control over inflammatory pathways, contributing to the chronic low-grade inflammation characteristic of cartilage aging. Future work will focus on quantifying histone acetylation levels (e.g., H3K9ac, H3K27ac) associated with key HDAC subtypes (HDAC2, HDAC5, HDAC11) in condylar cartilage across aging. This will help decipher how HDAC-mediated epigenetic regulation modulates the expression of cartilage homeostasis-related genes (e.g., COL2A1, aggrecan) and ultimately drives age-related structural deterioration—filling the current gap between HDAC expression patterns and their functional consequences.

This study has some limitations. First, HDAC expression was solely analyzed via IHC, factors like antibody titer, formaldehyde fixation duration, and decalcification time may have affected result reliability. Complement IHC with additional techniques—including in situ hybridization to detect HDAC RNA expression and Western blotting/immunofluorescence to quantify total HDAC protein levels—to further confirm HDAC expression patterns and eliminate artifacts from IHC. Second, while some time points were examined, the intervals between them might have been too broad to detect more subtle, yet potentially crucial, changes in HDAC expression during the aging process. Finer-grained sampling at more frequent intervals could reveal transient or short-lived expression patterns that are significant for understanding the dynamic nature of condylar aging. Third, we did not conduct a detailed investigation of differences between female and male mice, as it only presented histological and expression data from male mice in the main text despite confirming consistent expression patterns between genders in initial parallel analyses. Finally, the present study does not validate the mechanistic link between HDAC expression changes, histone acetylation, and cartilage homeostasis. Our results document correlations between HDAC dynamics and structural degeneration, but not causal relationships—this requires targeted functional experiments (e.g., histone acetylation measurements, HDAC knockdown assays) that build on the expression atlas presented here.

Conclusion

This study provides a spatial and temporal expression pattern of HDAC1-11 within the condylar cartilage during the period from postnatal to aging. It would be an important reference for understanding the spatiotemporal-specific expression of HDACs in the condyle. And it also may provide clues for regenerative strategies targeting TMJ developmental disorders. Further research into the role of these HDACs is, therefore, an essential next step in confirming their function and mechanism in the condylar cartilage.

Supplemental Material

sj-docx-1-car-10.1177_19476035251407304 – Supplemental material for Stage-Specific Expression of Histone Deacetylases in the Mouse Mandibular Condylar Cartilage

Supplemental material, sj-docx-1-car-10.1177_19476035251407304 for Stage-Specific Expression of Histone Deacetylases in the Mouse Mandibular Condylar Cartilage by Chubo Yang, Huishu Li, Jiaqi Kong, Mingxing Wang, Jingru Wang, Xinru Guo and Yuanbo Zhan in CARTILAGE

Supplemental Material

sj-docx-2-car-10.1177_19476035251407304 – Supplemental material for Stage-Specific Expression of Histone Deacetylases in the Mouse Mandibular Condylar Cartilage

Supplemental material, sj-docx-2-car-10.1177_19476035251407304 for Stage-Specific Expression of Histone Deacetylases in the Mouse Mandibular Condylar Cartilage by Chubo Yang, Huishu Li, Jiaqi Kong, Mingxing Wang, Jingru Wang, Xinru Guo and Yuanbo Zhan in CARTILAGE

Supplemental Material

sj-xlsx-3-car-10.1177_19476035251407304 – Supplemental material for Stage-Specific Expression of Histone Deacetylases in the Mouse Mandibular Condylar Cartilage

Supplemental material, sj-xlsx-3-car-10.1177_19476035251407304 for Stage-Specific Expression of Histone Deacetylases in the Mouse Mandibular Condylar Cartilage by Chubo Yang, Huishu Li, Jiaqi Kong, Mingxing Wang, Jingru Wang, Xinru Guo and Yuanbo Zhan in CARTILAGE

Footnotes

ORCID iDs

Jingru Wang

Yuanbo Zhan

Ethical Considerations

All experiments were conducted in accordance with relevant guidelines and regulations. This study was approved by the Ethics Committee of The Second Affiliated Hospital of Harbin Medical University (No. YJSDW2025 - 241).

Author Contributions

Chubo Yang: methodology, visualization, investigation, formal analysis, writing—original draft. Huishu Li: formal analysis and writing-review & editing. Jiaqi Kong: investigation, visualization, formal analysis, writing—original draft. Mingxing Wang: formal analysis and writing—review & editing. Jingru Wang: formal analysis and writing—review & editing. Xinru Guo: formal analysis and writing—review & editing. Yuanbo Zhan: conceptualization, experimental design, methodology, visualization, writing—original draft, writing—review & editing, funding acquisition. All authors gave their final approval and agreed to be accountable for all aspects of the work.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was partly supported by the Natural Science Foundation of Heilongjiang Province (grant number YQ2023H012) and the National Natural Science Foundation of China (grant number 82001488).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Supplemental material

Supplementary material for this article is available on the Cartilage website at .

References

Hatcher

. Anatomy of the mandible, temporomandibular joint, and dentition. Neuroimaging Clin N Am. 2022;32:749-61.

Ananthan

Pertes

Bender

. Biomechanics and derangements of the temporomandibular joint. Dent Clin North Am. 2023;67(2):243-57.

Iturriaga

Bornhardt

Velasquez

. Temporomandibular joint: review of anatomy and clinical implications. Dent Clin North Am. 2023;67(2):199-209.

Chen

Dutra

Mehta

O’Brien

Yadav

. Age-related changes in the cartilage of the temporomandibular joint. Geroscience. 2020;42(3):995-1004.

Chen

Wang

Luo

, et al. Age-related variations in position and morphology of the temporomandibular joint in individuals with anterior openbite and crossbite: a multi-cross-sectional comparative study. BMC Oral Health. 2022;22:200.

Silbermann

Livne

. Age-related degenerative changes in the mouse mandibular joint. J Anat. 1979;129(Pt 3):507-20.

Kitami

Kaku

Thant

Maeda

. A loss of primary cilia by a reduction in mTOR signaling correlates with age-related deteriorations in condylar cartilage. Geroscience. 2024;46(6):5995-6007.

Nickel

Iwasaki

Gonzalez

Gallo

Yao

. Mechanobehavior and ontogenesis of the temporomandibular joint. J Dent Res. 2018;97(11):1185-92.

Lopez-Otin

Blasco

Partridge

Serrano

Kroemer

. The hallmarks of aging. Cell. 2013;153:1194-217.

10.

Pal

Tyler

. Epigenetics and aging. Sci Adv. 2016;2:e1600584.

11.

Campisi

Kapahi

Lithgow

Melov

Newman

Verdin

. From discoveries in ageing research to therapeutics for healthy ageing. Nature. 2019;571(7764):183-92.

12.

Ukita

Matsushita

Tamura

Yamaguchi

. Histone H3K9 methylation is involved in temporomandibular joint osteoarthritis. Int J Mol Med. 2020;45(2):607-14.

13.

Liao

Sun

Liu

Jia

, et al. HDAC10 upregulation contributes to interleukin 1beta-mediated inflammatory activation of synovium-derived mesenchymal stem cells in temporomandibular joint. J Cell Physiol. 2019;234:12646-62.

14.

Liao

Sun

Wang

, et al. Histone deacetylase inhibitors attenuated interleukin-1beta-induced chondrogenesis inhibition in synovium-derived mesenchymal stem cells of the temporomandibular joint. Bone Joint Res. 2022;11:40-8.

15.

Gregoretti

Lee

Goodson

. Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis. J Mol Biol. 2004;338:17-31.

16.

Pasyukova

Symonenko

Rybina

Vaiserman

. Epigenetic enzymes: a role in aging and prospects for pharmacological targeting. Ageing Res Rev. 2021;67:101312.

17.

Patnaik

Pao

Zhao

Silva

Hylton

Chindavong

, et al. Exifone is a potent HDAC1 activator with neuroprotective activity in human neuronal models of neurodegeneration. ACS Chem Neurosci. 2021;12:271-84.

18.

de Ruijter

van Gennip

Caron

Kemp

van Kuilenburg

. Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J. 2003;370:737-49.

19.

Bhavsar

Ahmad

Adcock

. The role of histone deacetylases in asthma and allergic diseases. J Allergy Clin Immunol. 2008;121(3):580-4.

20.

Zhang

Yang

Zimmerman

Wang

Ross

Granger

, et al. CaMKII exacerbates heart failure progression by activating class I HDACs. J Mol Cell Cardiol. 2020;149:73-81.

21.

Hewitson

Dargan

Collis

Green

Moorjani

Ohri

, et al. Heart failure: the pivotal role of histone deacetylases. Int J Biochem Cell Biol. 2013;45(2):448-53.

22.

Schneider

Rasband

Eliceiri

. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671-5.

23.

Luder

. Age changes in the articular tissue of human mandibular condyles from adolescence to old age: a semiquantitative light microscopic study. Anat Rec. 1998;251(4):439-47.

24.

Rahmati

Nalesso

Mobasheri

Mozafari

. Aging and osteoarthritis: central role of the extracellular matrix. Ageing Res Rev. 2017;40:20-30.

25.

Lotz

Loeser

. Effects of aging on articular cartilage homeostasis. Bone. 2012;51(2):241-8.

26.

Loeser

Gandhi

Long

Yin

Chubinskaya

. Aging and oxidative stress reduce the response of human articular chondrocytes to insulin-like growth factor 1 and osteogenic protein 1. Arthritis Rheumatol. 2014;66(8):2201-9.

27.

Hong

Derfoul

Pereira-Mouries

Hall

. A novel domain in histone deacetylase 1 and 2 mediates repression of cartilage-specific genes in human chondrocytes. FASEB J. 2009;23(10):3539-52.

28.

Dovey

Foster

Cowley

. Histone deacetylase 1 (HDAC1), but not HDAC2, controls embryonic stem cell differentiation. Proc Natl Acad Sci U S A. 2010;107:8242-7.

29.

Chen

Zhang

Meng

Huang

Sheng

, et al. MicroRNA-455-3p modulates cartilage development and degeneration through modification of histone H3 acetylation. Biochim Biophys Acta. 2016;1863(12):2881-91.

30.

Bradley

Carpio

Westendorf

. Histone deacetylase 3 suppression increases PH domain and leucine-rich repeat phosphatase (Phlpp)1 expression in chondrocytes to suppress Akt signaling and matrix secretion. J Biol Chem. 2013;288:9572-82.

31.

Razidlo

Whitney

Casper

McGee-Lawrence

Stensgard

, et al. Histone deacetylase 3 depletion in osteo/chondroprogenitor cells decreases bone density and increases marrow fat. PLoS ONE. 2010;5:e11492.

32.

Bradley

Carpio

Olson

Westendorf

. Histone deacetylase 7 (Hdac7) suppresses chondrocyte proliferation and beta-catenin activity during endochondral ossification. J Biol Chem. 2015;290:118-26.

33.

Pham

Kaiser

Romsa

Schwarz

Gopalakrishnan

Jensen

, et al. HDAC3 and HDAC7 have opposite effects on osteoclast differentiation. J Biol Chem. 2011;286:12056-65.

34.

Vega

Matsuda

Barbosa

Yang

Meadows

, et al. Histone deacetylase 4 controls chondrocyte hypertrophy during skeletogenesis. Cell. 2004;119:555-66.

35.

Nishimori

Lai

Shiraishi

Kobayashi

Kozhemyakina

Yao

, et al. PTHrP targets HDAC4 and HDAC5 to repress chondrocyte hypertrophy. JCI Insight. 2019;4:e97903.

36.

Zhang

McKinsey

Chang

Antos

Hill

Olson

. Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy. Cell. 2002;110:479-88.

37.

Chang

McKinsey

Zhang

Richardson

Hill

Olson

. Histone deacetylases 5 and 9 govern responsiveness of the heart to a subset of stress signals and play redundant roles in heart development. Mol Cell Biol. 2004;24(19):8467-76.

38.

Kim

Lee

Ha Kim

Choi

Kim

. NFATc1 induces osteoclast fusion via up-regulation of Atp6v0d2 and the dendritic cell-specific transmembrane protein (DC-STAMP). Mol Endocrinol. 2008;22(1):176-85.

39.

Asagiri

Sato

Usami

Ochi

Nishina

Yoshida

, et al. Autoamplification of NFATc1 expression determines its essential role in bone homeostasis. J Exp Med. 2005;202:1261-9.

40.

Kim

Jin

Song

Youn

Lee

, et al. Negative feedback control of osteoclast formation through ubiquitin-mediated down-regulation of NFATc1. J Biol Chem. 2010;285:5224-31.

41.

Iorio

Laine

. What you may have missed in 2023: keeping up with the constant flow of new medical evidence. Ann Intern Med. 2024;177(suppl 5):S1-2.

42.

Blixt

Faulkner

Astleford

Lelich

Schering

Spencer

, et al. Class II and IV HDACs function as inhibitors of osteoclast differentiation. PLoS ONE. 2017;12(9):e0185441.

43.

Villagra

Cheng

Wang

Suarez

Glozak

Maurin

, et al. The histone deacetylase HDAC11 regulates the expression of interleukin 10 and immune tolerance. Nat Immunol. 2009;10(1):92-100.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.02 MB

0.00 MB

0.02 MB

4.96 MB