Introduction
As muscle and bone tissue is functionally and anatomically connected according to the mechanostat theory [
1], mechanical functional loading from masticatory system exhibits a crucial role in jaw growth and bone homeostasis [
2]. Although jaw bone can adapt to its functional environment [
3], the lack of a mechanical stimulus will result in alveolar osteopenia both in mandible and maxilla [
4]. Studies about the influence of masticatory muscles on craniofacial growth using various experimental animal models during adolescence and adulthood [
5–
9] consistently supported the concept that weak masticatory muscle functional changes reduce the functional capacity of jaw muscles and jaw muscle fibers [
10]. Consequently, mineralization and osteocyte density in mandibular alveolar bone both increase [
11,
12]. Low masticatory function also reduces the thickness of condylar cartilage and affects the balance between differentiation and proliferation of cartilage, thereby the decrease in condyle growth [
13,
14]. Condylar cartilage is defined as a secondary cartilage, which is more susceptible to mechanical loading than primary cartilage, such as femoral head cartilage and epiphyseal growth plates. Condylar cartilage is accompanied with chondrocytes and extracellular matrix (ECM) [
15]. Chondrocytes come from the mesenchymal cell differentiation and undergo a maturation process, including proliferation and hypertrophy. At the hypertrophic stage, the chondrocytes secrete ECM, which is mainly composed of collagens, oxytalan fibers, and glycosaminoglycans within proteogylcans; ECM sustains the mechanical properties of condyle [
16]. Various transcription and growth factors, including activator protein-1 (AP-1) transcription factor [
17], insulin-like growth factor-1 [
3], vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and transforming growth factor-b (TGF-b) [
18], are expressed in condylar chondrocytes and related to the amount of mechanical loading. However, systemic approaches for identifying the global changes of proteins in condylar cartilage in response to mechanical loading remain unclear. As proteins participate in all kinds of biological functions, they act as real functional molecules. Molecule expression at protein level can reflect actual conditions. In recent years, proteomic technology develops rapidly and is effective in detecting the global protein expression profiles under various physiological and pathological statuses. Proteomic level analysis of growth plate and articular cartilage in humans, mice, and rats provided detailed insights on the intracellular and extracellular proteome; this analysis also improves the understanding of chondrocyte maturation, ECM remodeling, and cartilage development [
19–
21]. Nevertheless, little information is known about protein expression using global quantitative proteomic analyses on condylar cartilage either in healthy or disease state. To our knowledge, only one study about the proteomic analysis of condylar chondrocytes in response to mechanical stress
in vitro was published [
22]. Li
et al. reported that six decreased proteins, including mitogen-activated protein kinase (MAPK) pathway inhibitor RKIP, heat shock protein GRP75, actin, and other cytoskeleton proteins, and an increased protein, that is, Rho-GTPase-activating protein, are involved in the early-response to mechanical stress in neonatal rat mandibular chondrocytes
in vitro [
22]. Further investigations are needed to study the loading signal transduction mechanisms in condylar cartilage
in vivo experimental protocols. The proteomic technique represents an important method as it can display many proteins simultaneously. Thus, this tool presents advantage over traditional methods (such as immunohistochemical (IHC) staining and Western blot technique) and can expand our understanding of the molecular basis of condylar mechanical transduction. Isotopic labeling and unlabeled methods are currently used for the relative comparison of protein expression under various physiological and pathological conditions [
23,
24]. Isobaric tag for relative and absolute quantitation (iTRAQ) is one of the most popular labeling methods and provides widely, useful insights on comparative proteomics [
25]. Combined with liquid chromatography and mass spectrometry (MS), iTRAQ can provide high-quality qualitative and quantitative data and quantify a larger number of ribosomal proteins, scarce proteins, and transcription factors than with traditional 2D electrophoresis [
26–
28].
In the present study, we selected the growing condylar cartilages from post-natal 16-day-old Sprague–Dawley rats (without any mastication) after a four-week soft-diet or hard-diet. Histological and tartrate-resistant acid phosphatase (TRAP) staining indicated the altered histological structure of condylar cartilage and increased bone remodeling activity in hard-diet group. Proteomic analysis using iTRAQ labeling technique on these two kinds of cartilage extracts identified 805 differentially expressed proteins that were non-redundant proteins with high confidence value. Comprehensive gene ontology (GO) annotations were also performed from GO and PANTHER databases for bioinformatics analysis. Collagen types I and II were further validated by immunohistochemical staining and Western blot analysis.
Material and methods
Animal and experimental design
Eighty 16-day-old, male SD rats were divided into two groups: one group was fed with whole pellet/hard diet, and the other group was fed with a soft, powdery food, as described previously [
17,
29]. The animals were raised under climate-controlled conditions: 55% humidity, a 12 h /12 h light/dark cycles, 24–26 °C room temperature, and free access to similar amount of water and food. All animals were sacrificed after four weeks.
Sample preparation
For proteomic and Western blot analysis, 70 condyle tissue samples from each group were stored in liquid nitrogen immediately, until all the cartilages were collected. In addition, 10 samples from each group were dissected, fixed in 4% paraformaldehyde for 48 h, demineralized in ethylene diamine tetra-acetic acid solution at 4 °C for 2–3 weeks, and finally embedded in paraffin wax (5 mm serial sagittal sections). The illustration of experimental procedure is shown in Fig. 1.
Histological staining
For histological observations, 5 mm-thick paraffin-embedded mandibular sections from both groups were prepared by following standard protocols for paraffin preparation and stained by toluidine blue- and safranin O (Sigma, St. Louis, MO, USA) staining. In addition, TRAP (Sigma, St. Louis, MO, USA) staining was performed to detect and quantify the number of osteoclasts at the juncture between condylar cartilage and subchondral bone. TRAP-staining positive cells in subchondral layer revealed numerous multinucleated cells attached to the bone at resorption lacunae.
Preparation of condylar cartilage proteins for proteomic analysis and iTRAQ labeling
Briefly, 40 condylar cartilages per group were mixed together, pulverized mechanically in liquid nitrogen, and lysed in a mixture of ice-cold RIPA lysis buffer (Beyotime, China). The crude extracts were vortexed vigorously on ice for 30 min at 10-min intervals and centrifuged at 12 000 r/min for 15 min at 4 °C. Protein concentration was measured by using BCA assay (Beyotime, China). Before labeling with iTRAQ reagent, 100 mg of protein per group was reduced, alkylated, digested, pooled together, and washed. Digested proteins were labeled with 4-plex iTRAQ reagents according to the manufacturer’s instructions (iTRAQ Reagent Multi-plex Kit, Absciex, Framingham, MA, USA). The samples from baseline (newborn rat condyles), soft-diet, and hard-diet groups were labeled with iTRAQ tags 114.1, 116.1, and 117.1, respectively. These samples were mixed before analysis. Subsequently, 10 ml aliquot of three samples was obtained, passed on a column by C18 ZiptipTM (Millipore, USA), and eluted by 90% ACN. Finally, the samples were concentrated by SpeedVac, and their labeling efficiency was evaluated by mass spectrum.
2D nano-HPLC and MALDI-TOF/TOF MS experiments
The labeled peptide mixture was purified and fractionated by 2D separation with 2D nano-HPLC (LC-20A, SHIMADZU, Japan). The samples were first diluted in strong cation exchange column, including Buffer A (10 mmol/L ammonium formate/0.1% formic acid) and Buffer B (500 mmol/L ammonium formate/0.1% formic acid). Subsequently, they underwent gradient elution, which started with 0% Buffer B and rose to 20%, 50%, and 100% Buffer B over 24 min. The peptides were resuspended in the second dimension using reverse-phase (RP) analytical capillary column (Thermo, Waltham, MA, USA), including Buffer A (5% acetonitrile/0.1 trifluoroacetic acid) and Buffer B (90% acetonitrile/0.1 trifluoroacetic acid). Afterward, peptides eluted from the capillary column were mixed at a 2 ml/min continuous flow of CHCA matrix solution.
The RP analytical column eluent was spotted onto target plates for MALDI-TOF-TOF-MS measurements with AccuSpot system (Shimadzu, Japan). All MS/MS spectra were identified by MASCOT software (version 2.1, Matrix Science, London, UK) and the rat SWISSPROT protein database (Release 2014_01). The searching parameters were as follows: mass spectrometry (MS)/MS tolerance±0.1 m/z and MS tolerance±0.15 m/z; allowance of missed cleavage, 1; mass spectra over the scanning m/z range of 800–4000 Da; fragmented ion tolerance, 0.2 Da; and consideration for variable modifications. The exclusion parameters were as follows: 80% confidence limit was applied to all reported data. Cut-off confidence values on accepting protein identification for Mascot were 80%. Only protein detected in each biological replicate was included; this protein must contain at least two distinct high-scoring peptides. iTRAQ ratios were analyzed automatically by GPS explorer (TM) v3.6 software. Quantification results for all proteins were considered as differentially expressed according to a threshold of iTRAQ ratio≥1.20 or≤0.80 [
30].
Bioinformatics analysis
The identified proteins were analyzed according to their molecular functions and biological processes; protein lists were uploaded as the official complete rat gene symbols from the web-based DAVID software (https://david.ncifcrf.gov/) [
31]. KEGG pathway (http://www.genome.jp/kegg/pathway.html) [
32] and PANTHER pathway databases (http://www.pantherdb.org/) analyzed the most remarkable signaling pathways of the differentially expressed proteins.
IHC staining
On the basis of our proteomic results, we screened out two important collagens, namely, types I and II, for further confirmation. For IHC staining, the unstained sections were deparaffinized and subsequently heated in a 1000 W microwave oven for antigen retrieval. Endogenous peroxidase activity was deactivated by incubation in 3% hydrogen peroxide for 20 min at room temperature. The slides were incubated with an anti-type I collagen antibody (Abcam, Ab34710/1:200 dilution, UK) and anti-type II collagen antibody (Abcam, Ab34712/1:200 dilution, UK) at 4 °C overnight. After incubation with a biotinylated secondary antibody for 30 min, the slides were washed in phosphate-buffered saline (PBS) for 5 min twice and then incubated with streptavidin-peroxidase-based complex for 15 min at room temperature. Finally, a 0.05 mol/L Tris solution of 0.05% DAB (Sigma, CA, USA) in 0.01% hydrogen peroxide was used for color reaction after 3 min at room temperature. The slides were dehydrated in gradient alcohol and xylene and counterstained with hematoxylin. Negative controls were incubated with PBS, instead of the primary antibody, to avoid nonspecific immunoreaction. Stain intensity of randomly selected slides between two groups was semi-quantified using Olympus microscope and digital camera (Olympus Bx51, CCD: Olympus DP71, Japan) and Image-Pro Plus 6.0 software (Media Cybernetics, MD, USA).
Western blot analysis
Western blot analysis of the proteins of collagen types I and II confirmed the results of iTRAQ analysis. Condylar cartilage tissue is pooled together from 30 samples of each group and frozen in liquid nitrogen. Subsequently, 200 ml RIPA lysis buffer (50 mmol/L Tris (pH 7.4), 1% Triton X-100, 150 mmol/L NaCl, 0.1% SDS, 1% sodium deoxycholate), 2 ml of phosphatase inhibitor cocktail (Roche, Germany), and 2 ml of PMSF (Sigma, USA) were added before grinding. The protein samples were spun down at 12 000 r/min for 10 min at 4 °C, extracted using a protein extraction kit (BioRad, USA), and quantified. For each blot, the protein extracts were fractionated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, USA). Transferred membranes were incubated in blocking solution with TBST buffer containing 5% w/v non-fat milk. They were also incubated overnight with primary antibodies, namely, anti-GAPDH antibody (Abcam, Ab8245/1:1000 dilution, UK), anti-type I collagen antibody (Abcam, Ab34710/1:500 dilution, UK), and anti-type II collagen antibody (Abcam, Ab34712/1:250 dilution, UK), in TBST (10 mmol/L Tris-HCL, 0.1% Tween-20, and 150 mmol/L NaCl) supplemented with 1% BSA at 4 °C. After hybridization with corresponding secondary antibodies from Cell Signaling, the membrane was visualized using ECL Western blot detection system (ECL kits, #170-5060, Bio-Rad, USA). The results were digitized using GE Image Quant LAS 4000 mini analyzer (GE Healthcare, Pittsburgh, USA).
Statistical analysis
All statistical data were processed with SPSS version 13.0 (SPSS Inc., Chicago, USA) and presented as mean±standard deviation (SD). The data were performed by one-way ANOVA, and t-test was subsequently performed. All differences were considered statistically significant at a P-value<0.05.
Results
Histomorphometry analysis of condylar cartilage
In 44-day-old rats, the cartilages stained with toluidine blue clearly presented three regions (anterior, superior, and posterior regions) [
33] and four layers (fibrous, proliferative, maturing, and hypertrophic layers) (Fig. 2A and 2B). Positive safranin-O staining was observed strongly in the hypertrophic layer, whereas no staining was found in the fibrous layer of condylar cartilage. Safranin-O staining was weaker in soft-diet group compared with that in hard-diet group. The anterior region was thinner in soft-diet group than in hard-diet group (
P<0.01) (Fig. 3A and 3B).
We counted the number of TRAP-positive cells below the hypertrophic layer of condylar cartilage, where endochondral ossification occurred. The number of TRAP-positive cells in the hard-diet group increased compared with the soft-diet group (P<0.01) (Fig. 3A and 3C), which implied an increase in bone remodeling activity. All the above observations confirmed that this model was suitable in identifying proteins involved in mechanical functional changes in condyles.
Differentially expressed protein identification, quantification, and GO analysis
iTRAQ analysis of differentially expressed proteins in the rat condylar cartilage induced by reducing masticatory force was conducted. The most highly expressed identified protein was a calcium ion-binding protein, known as cadherin, or EGF LAG seven-pass G-type receptor 3 precursor encoded by
CELR3 gene. Among a total of 805 identified proteins, we performed GO analysis with three main aspects: cellular component, molecular function, and biological process. GO biological process analysis revealed that a significant proportion of proteins were involved in metabolic process (51.8%), cellular process (47.7%), biological regulation (27.5%), localization (19.6%), developmental process (16.6%), and response to stimulus (15.2%). In terms of molecular function, most of these proteins functioned in catalytic, binding, transporter, receptor, structural molecular, and nucleic acid binding transcription factor activities (Fig. 4). According to PANTHER database, the identified proteins were categorized into receptors (14.2%), nucleic acid binding proteins (12.9%), hydrolases (12.0%), transporters (10.6%), and enzyme modulators (8.6%). PANTHER protein class analysis of our selected data set is shown in Table 1. The main classes were the following: calcium-binding proteins, cytoskeletal proteins, ECM proteins, signaling molecules, and transcription factors, some of which were recognized in other studies [
20].
Some remarkable proteins were obtained from the hard-diet group. Many upregulated proteins were related to cytoskeletons (actin, myosin-10, formin-binding protein 1-like, chloride intracellular channel 6, dynamin-1/2, tubulin α-3 chain, and calponin-1), ECM proteins (type I/II collagen, integrin, chordin, and laminin), loading-induced growth factors (VEGF, FGF, and TGF-b), transcription factors (transcription factor Maf and fos-related antigen 1), and cytokines (tumor necrosis factor ligand superfamily member, interleukin-33 precursor, and interleukin-1 receptor-like 2 precursor) (Table 1). Among these proteins, GO annotation showed that VEGF was located in extracellular region. VEGF participated in angiogenesis and vasculature development. VEGF also activated cytokine–cytokine receptor interaction, mTOR signaling pathway, and focal adhesion. TGF-b activated MAPK signaling pathway, Wnt signaling pathway, and cytokine–cytokine receptor interaction according to KEGG pathway analysis. Transcription factors, namely, transcription factor AP-1 and 14-3-3 protein β/α (protein kinase C inhibitor protein 1), were downregulated compared with the soft-diet group. The molecular function of 14-3-3 protein was associated with transcription factor binding and transcription repressor activity; 14-3-3 protein is also a key mediator in many signal transduction pathways.
KEGG and PANTHER pathway analyses
KEGG pathway analysis suggested that the most prominent and relevant pathways involved in mechanical stress changes included calcium signaling pathway (
P = 6.73E-09), gap junction (
P = 0.013), ErbB signaling pathway (
P = 0.016), and MAPK signaling pathway (
P = 0.035) (Table 2). In the calcium signaling pathway, receptor tyrosine-protein kinase erbB-2/3 precursor (encoded by
ERBB2 and
ERBB3 gene), phospholipase C-γ-1 (PLCB1), phospholipase C-γ-2 (PLCB2), protein kinase C β type (KPCB), and protein kinase C γ type were upregulated in response to mechanical stress; these proteins were also involved in ErbB signal pathway. Voltage-dependent P/Q-type calcium channel subunit α-1A (encoded by CAC1A gene), voltage-dependent T-type calcium channel subunit α-1G/H/I (CAC1G, CAC1H, and CAC1I), and KPCB interlinked with the MAPK signaling pathway. Three abundant related-signaling pathways, namely, FGF signaling pathway, angiogenesis, and heterotrimeric G-protein signaling pathway, were also enriched in condylar cartilage by PANTHER pathway analysis (Table 3); the latter two pathways were described in literature during the mechanotransduction process within condyle [
34–
36].
Validation of alterations in collagen types I and II by IHC and Western blot
Collagen types I and II are protein markers that can detect chondrogenic differentiation. In the current study, type I collagen was mainly absent in maturing and hypertrophic layers, although it is present throughout the condylar cartilage layers [
37]. Type II collagen, the major component of the condylar cartilaginous matrix [
35], was observed in maturing and hypertrophic layers. In the soft-diet group, type I and type II collagen-positive cells decreased compared with the hard-diet group (Fig. 3A and 3D). The negative controls exhibited no immunoreactivity. As shown in Fig. 5, the band for GAPDH was clearly detected in both groups. Western blot results revealed that the protein expression of type I and type II collagens decreased in soft-diet group compared in the hard diet group. The results were in agreement with current IHC and iTRAQ data, which could confirm the proteomic analyses based on iTRAQ.
Discussion
In the current study, the timing of the whole experiment was selected according to the basis of rat molar functional occlusion. With the eruption of the first (25th day), second (28th day), and third molar teeth (40th day) [
38], the teeth begin to transmit masticatory forces to the mandible through periodontal ligament (PDL). The condylar cartilage is mainly subjected to compression forces when the jaw-closing muscles contract. As the tooth-PDL-alveolar bone complex acts synergistically in response to the initial loads during mastication [
39], occlusal forces directly influence the jaw alveolar bone macro- or microstructure; furthermore, condylar cartilage is sensitive to the alterations in occlusal loading during occlusal development stage [
38,
40]. These observations were the reason why the present rat age point and designed experimental period were used.
Three major findings were obtained from the present study. First, we confirmed that low masticatory demands significantly affected the histological structure of condylar cartilage. In the soft-diet group, collagen fiber contents decreased. In addition, a thinner anterior region of the condyle and a weaker safranin-O staining (associated with chondrocyte-like cells and the evidence of condyle remodeling) were observed in the soft-diet group than in the hard-diet group; the results were in agreement with previous findings on growing rodents [
41]. Sufficient loading exhibits an important role in maintaining ideal chondrocyte proliferation and matrix production [
38].
Second, among all the differentially expressed proteins identified by iTRAQ, various altered proteins were connected with the cytoskeleton, ECM protein, loading-induced growth factors, transcription factors, and cytokines during the mechanotransduction process in condylar cartilage. In the ECM of condylar cartilage, collagens and proteoglycans mainly interact with each other and build various architectural networks [
42]. In particular, types I and II collagens are two representative markers for condylar cartilage. In our study, the number of Col I and II immunopositive cells in the hypertrophic layers increased in the hard-diet group; this result was consistent with the histological results that showed a thicker hypertrophic layer of the condylar cartilage in hard-diet group than in soft-diet group (Fig. 3A). In addition, other ECM proteins, including CO5A1, MEGF6, ITB4, CSPG2, CSPG2, FETUA, CHRD, LAMB2, and AP3M1, were upregulated when the masticatory force increased (Table 1). Hard diet induced matrix production and affected endochondral ossification of condylar cartilage in rodents.
Proteomic analysis also identified several members of the VEGF family. VEGF regulates chondrocyte metabolism, vascular development, and angiogenesis; it is also essential for ECM remodeling and endochondral ossification in the condyle [
34]. Consistent with previous study [
43], our study found that VEGF protein expression could be promoted by mechanical loading, which indicated that VEGF has an important role in condylar cartilage remodeling. Recently, TGF-b/bone morphogenetic protein (BMP) signaling is required for maintaining homeostasis of cartilage-bone unit because of a link between mechanical signaling and TGF-b or BMP expression [
18]. However, small amount of data are available on the connection between mechanics and TGF-b or BMP signaling pathway in the condylar tissue. TGF-b1 is highly expressed in mature and degenerated layers of condyle [
44]. TGF-b signaling regulates condylar cartilage proliferation and differentiation [
45]. In our current study, TGF-related proteins (encoded by
TGFR-3,
TGFR-1,
WDR7, and
WISP2 genes) increased slightly in the hard-diet group, whereas BMP-3 slightly decreased compared with the soft-diet group. Further investigation is required to validate the proof of existence of an intermediate crosstalk between TGF-b/BMP signaling and mechanotransduction in condylar cartilage. The 14-3-3 protein β/α or protein kinase C inhibitor protein 1 encoded by
1433B gene was downregulated in the hard-diet group, which was not reported previously in condylar cartilage. This kind of intracellular proteins presented several isoforms. Furthermore, soluble 14-3-3e proteins released by loaded mouse osteoblasts/osteocytes are involved in cartilage degradation in osteoarthritis [
46].
Remarkably, the dentin sialophosphoprotein precursor (DSPP) in the extracellular region was significantly upregulated in the hard-diet group compared with the soft-diet group. DSPP, a member of small integrin binding ligand, N-linked glycoprotein family, is expressed in tooth, cementum, bone, and some non-mineralized tissue, such as condyle [
47]. Mice models without DSPP exhibited decreases in the amount of condylar cartilage, which suggested the necessity of DSPP for postnatal condylar cartilage growth and maintenance [
42]. Our investigation indicated the potential function of DSPP in mechanotransduction of condyle. All these findings demonstrated a global view that altered proteins are associated with reduced masticatory function.
Third, most peptides were labeled with intact information from protein post-translational modification by using iTRAQ coupled with nano-HPLC and MALDI-TOF/TOF technology. Several signaling pathways were involved within condylar cartilage induced by reducing dietary loading. Calcium signaling pathway was the most notable signal pathway in our study. Ca
2+ could exert various biological effects, such as the activation of MAPKs pathways [
48]. In the current study, many represented proteins related with calcium signaling were interlinked with ErbB and MAPK signaling pathways on the condylar cartilage with altered functional mastication loading (Table 2). Clearly, a significant crosstalk existed among different pathways by the activity of multiple signaling proteins under condylar cartilage remodeling. MAPK signaling pathway is crucial for various phases of endochondral ossification and mechanoresponsiveness [
48]. In the hard-diet group, Ras GTPase-activating protein 1, GTPase NRas precursor, MAPK kinase kinase 12, and death domain-associated protein 6 involved in classical, JNK, and p38 MAP kinase pathways were upregulated. JNK, ERK, and p38/MAPK in condyle were not completely investigated; thus, a large amount of studies are necessary to understand the function of MAPK pathway in condylar cartilage mechanotransduction. In the hard-diet group, the most represented proteins related with Wnt signaling, such as axin-1, frizzled-5 precursor, protein kinase C γ type, Fos-related antigen 1, and Rho-associated protein kinase 1, increased with induced dietary loading. Other
in vivo and
in vitro studies indicated that Wnt signaling, together with its multiple interplays with other pathways, allows cartilage or subchondral bone to sense [
48] and relates to cartilage physiology and pathology [
49].
Nevertheless, the current study showed some limitations. Protein extraction from cartilage tissue is considerably difficult. Condylar cartilage comprises scattered chondrocytes and largely ECM; dominant aggrecan and collagen fibers within ECM may overwhelm the other protein signals. Some researchers combined physical disruption and chemical extraction to improve the efficiency of cartilage extraction [
50]. In our study, we pulverized the liquid nitrogen-frozen condylar cartilages to enhance extraction efficiency. Large amounts of collagen and proteoglycans were extracted via mechanical grinding of cartilages into powder; therefore low-abundance proteins can be hardly detected. iTRAQ method presents several disadvantageous features, such as labeling inefficiency and susceptibility to errors during precursor ion isolation [
51]. Additional molecular and biochemical experiments are needed to improve proteomic studies.
In conclusion, we identified differentially expressed proteins and various putative signaling pathways within condylar cartilage in response to reduced masticatory function induced by changing dietary consistency during adolescence. In addition, we hypothesized the detailed mechanisms by a quantitative comparative proteomic technique combined with further bioinformatics analysis. Condylar remodeling is a physiologically complicated process. Although the underlying molecular mechanisms during this process are still incompletely understood, new proteomic methods are available. These new methods are effective in determining a global perspective of various altered proteins and previously unrecognized biological process pathways in protein level.
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