Normal static loading is essential for the growth of the temporomandibular joint (TMJ) [
In the condylar cartilage, the interaction of parathyroid hormone related protein (PTHrP) and Indian hedgehog protein (Ihh) is considered to be a crucial biomechanical regulator to mediate the proliferation and differentiation in cartilage growth [
Providing a soft diet is a conventional method to decrease occlusal loading, based on an animal study [
The biomechanical strength in the maxillofacial region mainly depends on the masticatory muscles, and the muscle fibers are the most important contributor to the bite force. Most studies have focused on structural remodeling after functional changes and seldom evaluated muscular hypofunction. In this study, to evaluate the masseter and condyle in combination, we attempted to estimate the association between functional suppression and tissue degradation by examining muscle atrophy and structural remodeling factors after 4 weeks of decreased occlusal loading.
Sixty 5-week-old female Sprague-Dawley rats, weighing 180-200g, were used in this study. The animal experiment protocol was approved by the Medical Ethics Committee of the Hospital of Stomatology, Wuhan University. The animals were kept in a dedicated animal holding facility under veterinary supervision in SPF Animal Laboratory of School of Stomatology, Wuhan University. All animals were allowed free access to water and food.
Rats were randomly assigned into the following 3 groups: the control group (n=20), soft diet group (n=20), and BTX group (n=20). The rats were anesthetized with intra-abdominal injections of sodium pentobarbital (Cat:AS1090, Aspen Technology Inc., USA) at a dose of 50 mg/kg body weight. A 0.5-1cm incision was made on the bilateral buccal skin to expose the masseter. For the BTX group, 2-unit Botox (Allergan, Irvine, California; the solution was diluted with 1 unit for every 0.1 ml saline) solutions were intramuscularly injected into both sides. For the control and soft diet groups, the equivalent volume saline solution was injected. The incisions were then sutured with absorbable materials. For the control group, the regular consistency pellet was offered. For the SD and BTX groups, the pellet was blended into powder to minimize the hardness. It should be noted that, after BTX injection, the rats were unable to chew the regular or hard consistency diet; accordingly, the soft diet was then provided. The rats were sacrificed with an overdose of sodium pentobarbital after 4 weeks of treatment.
The specimens were dissected from the corpse, including the masseter muscles and mandibular bone. The masseter muscles were weighed using an electronic balance and the condylar cartilage was measured by electronic caliper. To expose the condylar contour, the TMJ capsule and adjunctions were completely dissected from the condylar head. The measurement of condylar head included the length, width, and thickness (the thickness was calculated by histological analysis).
After being weighed, the masseter muscles were fixed in 4% paraformaldehyde (PFA) solution at room temperature for 24 hours. The muscles were then transected in the central part to expose the cross-sectional area (CSA). After being embedded in paraffin, 4-
The immunohistochemical examination was performed according to the instructions of the manufacturer. The sections were incubated with primary antibodies against atrogin-1 (1:100 diluted in PBS buffer; GTX47819, GeneTex Inc.) and MuRF-1 (1:100 diluted in PBS buffer, IMX-3924, Novus). For negative controls, the primary antibodies were omitted. The 3,3′-diaminobenzidine (DAB-0031/1031, Maxim, Wuhan China) staining was used to detect the reaction. The sections were counterstained with hematoxylin and observed using a computer-assisted image-analyzing system.
Mandibular samples were decalcified in 10% EDTA for 4 weeks and then resected and stained with H-E and toluidine blue (Cat: G1032, Goodbio technology Co., Wuhan China) to determine the morphology and proteoglycan content of the condylar cartilage. The thickness of the central 1/3
The immunohistochemical analysis of the cartilage was performed as mentioned previously. The primary antibodies were PTHrP (1:200, SC-20728, Santa Cruz Biotechnology, Inc.), Ihh (1:250, AB-52919, Abcam), and Col 2
The TRAP staining (386A-1KT, Sigma-Aldrich, Inc.) was used to determine the osteoclast content in the subchondral bone. The procedure was performed according to the manufacturer’s protocol.
The muscle and cartilage were, respectively, detached from the fascia under microscopy and preserved in liquid nitrogen immediately. The tissues were then grinded in the RIPA lysis buffer (Cat:AS1004, Aspen Technology Inc., USA) supplemented with protease inhibitor cocktail (Cat:AS1005C, Aspen Technology Inc., USA) and PMSF (Cat:AS1006, Aspen Technology Inc., USA). When the disruption was completed, samples were centrifuged at 4°C, 12000 rpm for 5 min. The supernatant was collected and used as the total protein sample.
The BCA Protein Assay (Thermo Fisher Scientific) was used to determine the total protein levels. After being diluted with sample buffer, 10
To examine the subchondral bone, the condylar process was scanned by micro-CT (Y. Cheetah, X. YLON, Germany). The X-ray tube was set at 80 kv, 50 mA, and the scanned projection was 450 with an integration time of 0.6s and a resolution of 5
The representative samples from each group were randomly selected, and the H-E muscle section (x100 magnification) was calculated by ImageJ 1.50i (National Institutes of Health, USA). The value shown was used as the CSA of the masseter muscle.
Immunohistochemistry semiquantitative analysis considered both the staining intensity and positive proportion. The positive reactions were defined as brown staining in chondrocytes and matrix, and the counts were calculated by positive cells/total number. The intensity was scored as follows: 0, negative; 1, weak; 2, moderate; 3, strong. The proportion was scored as follows: 0, less than 5%; 1, 5%-25%; 2, 25%-50%; 3, 50%-75%; 4, more than 75%. The immunohistochemical evaluation index was calculated by multiplying the intensity and proportion. Based on a score ranging from 0 to 12, the value of 0-7 was considered as low expression and a value of 8-12 was considered as high expression.
The TRAP staining was estimated by counting the positive cells, which were red-wine colored with multiple nuclei. The observational regions were defined as the subchondral bone adjacent to the cartilage's border.
Nonparametric one-way analysis of variance (ANOVA) with
Body weight showed no significant difference among three groups. The contour surveying method is shown in supplemental figure (available
| | | ||||
---|---|---|---|---|---|---|
| | | | | | |
| 1.19 g | ±0.05 | 1.09 g | ±0.06 | 0.59 g | ±0.08 |
| 1.53 mm | ±0.07 | 1.46 mm | ±0.10 | 1.38 mm | ±0.18 |
| 3.76 mm | ±0.20 | 3.74 mm | ±0.19 | 3.38 mm | ±0.34 |
| 178.33 | ±30.96 | 139.13 | ±29.40 | 80.99 | ±10.29 |
Based on H-E staining, reduced CSA was observed in the BTX group (versus control, p<0.001; versus SD, p<0.05) (Figures
Histological changes and atrogin-1/MuRF-1 immunohistochemical staining of masseter muscles in the control, SD, and BTX groups. After 4 weeks of treatment, H-E staining of masseter muscle revealed an obvious atrophic condition in the BTX group, whereas no difference in the SD group was seen (a–c, j). The distinctive expression of atrogin-1/MuRF-1 could be easily found in the BTX group compared to that in the control and SD groups (d–i) (
On immunohistochemical analysis, semiquantitative analysis of atrogin-1/MuRF-1 revealed a low expression in the SD group, with almost 20% upregulation versus control (p<0.05), whereas a high expression in the BTX group, with more than 75% versus control (p<0.001), was seen (Figures
A nearly triple upregulation of atrogin-1/MuRF-1 in the BTX group versus control group (p<0.001, p<0.001) and SD group (p<0.001, p<0.001) was observed. The SD group had a moderate increase in atrogin-1 and doubling in MuRF-1 compared with that in the control was seen (p<0.05, p<0.001) (Figures
The protein fold change of atrogin-1/MuRF-1 after 4 weeks of treatment. Atrogin-1/MuRF-1 proteins were detected by western blot; the grayscale ratio to GAPDH was calculated (a). Compared with that in the control, the SD group had a double upregulation in MuRF-1 and a moderate enhancement in atrogin-1; the BTX group had a nearly triple fold increase in atrogin-1/MuRF-1 (b, c) (
No significant difference in cartilage length or width was observed between the control and SD groups; however, there was an obvious decrease in thickness. The BTX group showed a large decrease in width, length, and thickness compared to those in the control group (Table
H-E staining of the SD and BTX groups showed a notable decrease in cartilage thickness (SD versus control, p<0.001; BTX versus control, p<0.001) (Table
Condylar cartilage morphology. H-E staining illustrated a decreased cartilage thickness and decreased chondrocyte number (a–c). Compared to that in the control group, the experimental groups demonstrated a trend of condensation and reduction in cellular number (d–f). Toluidine blue staining in the SD and BTX groups revealed a faint chromatosis compared to that in the control, which might indicate a decrease in the synthesis of proteoglycans (g–i) (
Immunostaining was performed to determine the expression of PTHrP, Ihh, and Col2a1. The results showed that PTHrP and Ihh positive staining was concentrated within the proliferative and prehypertrophic layers. The PTHrP positive chondrocytes of the control group were predominantly more abundant than those in the SD and BTX groups (p<0.01, p<0.001) (Figures
Immunostaining changes of condylar cartilage. The PTHrP positive cells were distributed in the proliferative and early hypertrophic layers (a–c). Ihh was expressed in the extracellular space and concentrated within the proliferative layer (d–f). The Col2a1 positive area was shown in the hyperplastic layers; the intensity and area predicted the matrix's volume (g–i) (
Western blotting assessment of cartilage demonstrated a pronounced change in the protein expression levels of PTHrP, Ihh, Col2a1, and Col X (Figure
Protein fold change in cartilage after treatment. Western blot analysis of condylar cartilage. PTHrP and Ihh showed a similar declination after decreased occlusal loading treatment (b, c). The two extracellular collagens, Col2a1 and Col X, were more sensitive to the altered loading, which resulted in a dramatic decrease in the BTX group (d, e) (
Micro-CT tests were performed as described previously, and the 3D reconstructions are illustrated in Figures
Subchondral bone loss. The selected areas were isolated from the condylar head, which aimed to reserve the trabecular bone to avoid interference by the cortical bone (a–c). The isolated spongy bones were reconstructed as illustrated (d–f). The changes of BV/TV, Tb.N, Tb.Th, and Tb.Sp evidenced osteopenia that occurred in the subchondral bone (g–j). TRAP staining showed that the osteoclasts (black arrows pointed) were significant upregulated in the BTX group compared with that in the control and SD groups (k–m) (
Tartrate-resistant acid phosphatase (TRAP) staining was performed to determine osteopenia in the condylar subchondral bone (Figures
It is widely accepted that the tissues are able to remodel in accordance with different levels of activity [
Initially, we estimated muscular function through muscle mass assessment, CSA, and atrophic factors (atrogin-1/MuRF-1) after 4 weeks of treatment. This is an objective indication to evaluate the suppressed muscle performance and strength. Studies have shown that the CSA and atrophic factors are an objective indication to evaluate muscle function [
Thereafter, we determined changes of condylar cartilage according to such decreased loading. The contour measurements of the condylar head manifested a degenerative reconstruction, and the cartilage’s thickness was more vulnerable to degradation than the width and length after unloading treatment [
As reported by other scholars, subchondral bone, cortical bone, alveolar bone, and even the growth of the mandible were influenced by loading as well [
From previous results, the increased expression of atrophic protein in muscles was inversely related to the biomechanically sensitive factors in condylar cartilage. Additionally, when the amount of atrophy exceeded the metabolic balance, the mechanical loading would be largely suppressed. Under such a situation, the degraded remodeling would not be limited to the cartilage; it would also demonstrate in the subchondral bone.
Biomechanical therapy is one of the most noteworthy treatments to reconstruct the relevant tissue [
In recent years, botulinum toxin injection became popular in plastic therapy, especially for masseteric hypertrophy [
A systemic review has showed that the BTX intra-articular injection could be effective in relieving the pain due to osteoarthritis [
This work was supported by grant from the Wuhan Science and Technology Bureau (no. 2015061701011641).
The authors declare no conflicts of interest related to the study.
Supplemental Fig: the measurement of condyle and the specimen. The condylar head was measured by electronic caliper (A, B). After 4 weeks of treatment, the specimens were dissected from the corpse. Regarding the BTX group, a reduced condylar area was observed (C, D).