A histochemical study on condylar cartilage and glenoid fossa during mandibular advancement
Abstract
Objective:
To evaluate cellular hypertrophic activities in the mandibular condylar cartilage (MCC) and the glenoid fossa (GF) during mandibular advancement in the temporomandibular joint (TMJ) of Sprague-Dawley rats, as evidenced by fibroblast growth factor 8 (FGF8).
Methods and Materials:
Fifty-five female 24-day-old Sprague-Dawley rats were randomly divided into four experimental and control groups, with a mandibular advancement appliance on the experimental rats' lower incisors. The rats were euthanized on days 3, 14, 21, and 30 of the study, and their TMJ was prepared for a immunohistochemical staining procedure to detect FGF8.
Results:
FGF8 expression was significantly higher among the experimental rats (P = .002). Patterns of ascension and descension of FGF8 expression were similar in experimental and control samples. The results show an overall enhanced osteogenic transition occurring in both the MCC and the GF in experimental rats in comparison with controls. The level of cellular changes in the MCC is remarkably higher than in the GF.
Conclusion:
In the MCC and the GF, cellular morphologic and hypertrophic differentiations increase significantly during mandibular advancement. It is also concluded that endochondral ossification in the MCC and intramembranous ossification in the GF occur during adaptive remodeling.
INTRODUCTION
Several studies have discussed mandibular advancement as a functional therapy for skeletal Class II malocclusion1 and have shown that a fundamental factor in regulating cellular activities during tissue morphogenesis is mechanical stress. Forward positioning of the mandible is followed by adaptive remodeling in the mandibular condylar cartilage (MCC) and the glenoid fossa (GF).2–6 Growth modification of the lower jaw during mandibular forward positioning is a successful example of bone remodeling in response to a change in the biophysical environment.1 Many studies with rats and monkeys have shown that new bone formation in the condyle and the GF occurs in response to mandibular advancement.6–8
This remodeling occurs by expression of endogenous regulatory factors of cells in the mandibular condyle through an endochondral ossification process1–3 and intramembranous ossification in the GF.4,6,7
The population size of the mesenchymal cells present in the subperiosteal connective tissues of the MCC directly affects the number of bone-making cells available to engage in the formation of new bone during craniofacial development. Undifferentiated mesenchymal cells in the extracellular matrices (ECMs) give rise to other cell types as the need arises and are present in the ECM of developing bones in the skull, including the temporal bone.9
The fibroblast growth factors (FGFs) regulate mesenchyme and chondrocyte proliferation in MCC adaptive remodeling.10 FGF signaling results in a decrease in chondrocyte proliferation and an acceleration of hypertrophic differentiation and morphologic changes in chondrocytes.5 FGF8 is expressed in highly proliferating, columnar chondrocytes and in early hypertrophic and hypertrophic chondrocytes.11
The aim of this study is to evaluate hypertrophic activities in the MCC and the GF during mandibular advancement in Sprague-Dawley rats, as evidenced by FGF8. In this study, FGF8 is used as an indicator of cellular chondrogenic and morphologic differentiation and hypertrophic activity, to demonstrate the histochemical nature of the adaptive response of bone to mandibular protrusion.
MATERIALS AND METHODS
Fifty-five female Sprague-Dawley rats at the age of 24 days were randomly divided into the experimental group (n = 35) and the control group (n = 20); the study was approved by the Westmead Animal Ethical Committee (Protocol No: 4113.06-08). All rats were kept in the same well-controlled temperature and humidity environment. They were fed a soft palate diet and had uninhibited access to water 24 hours a day throughout the entire experimental period (Table 1).
The animals were sedated and a crown former was positioned on their lower anterior incisors in such a way that it caused mandibular forward-downward positioning during the rats' rest and functional bite (Figure 1B). Animals in the control groups were not fitted with an appliance and were untreated (Figure 1A). Body weight was monitored throughout the experiment.
Citation: The Angle Orthodontist 81, 2; 10.2319/021710-99.1
Euthanasia and Tissue Preparation
The animals in each subgroup were euthanized, respectively, by carbon dioxide gas (Aligal 2, Air Liquide Australia Ltd, Fairfield NSW, Australia) on the 27th, 38th, 45th, and 54th days of the rats' age.
Immediately after death, the heads were removed and were fixed in 4% paraformaldehyde for 24 hours. The heads were then decalcified in 20% ethylenediaminetetraacetic acid (EDTA), pH 7 to 7.4, at 4°C to 8°C, for 4 to 6 weeks.
The temporomandibular joint (TMJ) was dissected, and surrounding soft tissues were removed until the TMJ was exposed. Excess tissues were removed, and specimens with the buccal surface of the ramus parallel to the surface of the block were embedded in paraffin. Serial sections 5 µ thick were cut through the TMJ at the parasagittal plane using a rotary microtome (Leitz 1516, Leica Microsystems, Wetzlar, Germany); sectioning was continued until the approximate middle of the condyle was reached. At this level, a few sections were floated onto glass slides coated with poly-L-lysine.
Individual variations occurred in TMJ orientation in the skull. Thus, to make a reliable comparison, the plane of each section throughout each of these anatomic variables was adjusted as identically as possible between samples. Sections cut from each sample were assigned to immunohistochemical staining for fibroblast growth factor 8 (FGF8).
Immunohistochemical Examinations
The specific primary antibody used was FGF8 goat polyclonal antibody collagen (N-19, Cat #SC 6958, Lot #E300, 200 µg/mL; Santa Cruz Biotechnology Inc, Santa Cruz, Calif). The secondary antibody was rabbit antigoat immunoglobulin (Ig)G (HRP, Code No. P0449; Dako A/S, Glostrup, Denmark).
Immunohistochemistry was carried out using a method in which the sections were dewaxed and rehydrated and were treated with glycine, 3% hydrogen peroxide, horse serum, the primary antibody, and the secondary antibody.
Then the slides were dipped in 3,3′-diaminobenzidine (DAB) in chromogen solution (Dako Liquid DAB + Substrate Chromogen System, Code K3467, Dako A/S) and finally were counterstained with Mayer's hematoxylin for background staining. Negative controls were included, in which the primary antibody was replaced by FGF8-blocking peptide (N-19 P, Cat #SC 6958 P, Lot #F268, 100 µg/0.5 mL, Santa Cruz Biotechnology) to ascertain the specificity of the immunostaining.
After the clearing protocol, slides were covered by mounting medium (Fisher Scientific Permount SO-P-15, 500 mL, 1.1 pt; Fisher Scientific, Fair Lawn, NJ) and a coverslip for long-term storage of slides and additional microscopic studies.
Quantitative Imaging
Digital images were taken from stained tissues with a Leica digital imaging microscope and its software (Leica Application Suite Software; Leica Microsystems, Bannockburn, Ill) at 10×, 20×, and 60× magnification.
Expressions of FGF8 were quantified by manually counting the cells of positive reacted immunostaining signals on the computer screen from 20× magnified images. Cells were counted from the middle quarter of the MCC and the distal third of the GF, where the most prominent cellular responses to mandibular repositioning occur.2,12 Cells that were stained with certain intensity were counted, and those that were weakly stained were excluded.
Statistical Analysis
After the first counting, data were collected again 4 weeks later by the same observer, and the method of error (ME) was tested. No statistically significant difference was noted among the registrations. Data were analyzed using a statistical package (Statistical Package for the Social Sciences [SPSS] for Windows, version 16.0, SPSS Inc, Chicago, Ill).
RESULTS
The cytoplasms of early hypertrophic and hypertrophic cells, beneath the layer of cell proliferation and above the erosive zone, were positively stained for FGF8 (Figure 2D,G). FGF8 is located mainly in the cytoplasm of osteochondroprogenitor cells, chondroblasts, and chondrocytes before their degeneration, which is shown by extra magnification of a typical immunopositive cell for FGF8 from the hypertrophic layer of the mandibular condylar cartilage of a 38-day-old experimental sample (Figure 2).
Citation: The Angle Orthodontist 81, 2; 10.2319/021710-99.1
The level of FGF8 expression in the condyle and glenoid fossa of experimental rats generally is significantly higher than in relevant control samples. Furthermore, it is clear that the amount of cellular activity in the mandibular condylar cartilage is greatly higher than in the glenoid fossa in both controls and experimental rats at different stages of growth and development. The effect of the appliance on FGF8 expression is generally significant in the MCC and the GF (FGF8c, P = .002; FGF8gf, P = .002). The level and pattern of FGF8 expression are shown in Table 2 and Figure 3.
Citation: The Angle Orthodontist 81, 2; 10.2319/021710-99.1
DISCUSSION
The histologic structures of the rats' TMJ are similar to those of humans, with morphologic differences.13 Because of this similarity and the possibility of a histochemical study on rats based on previous studies,14,15 55 Sprague-Dawley rats were used in this experiment, as in other histologic and biochemical investigations.3,4,8,12–14,16,17
Even though some studies on monkeys indicate that such adaptive responses are nonexistent and negligible,18 several other findings, such as those of the current study, indicate positive significant TMJ adaptation in response to mandibular advancement.2,3,16,19–22 However, the pattern of this adaptive response in the MCC is different from that in the GF.
The adaptive response of the MCC-GF complex could be described by the growth relativity hypothesis and the functional matrix theory. The mandible is displaced to a forward position, viscoelastic forces are applied on the MCC-GF complex at the same time in reverse directions, and the forces are transduced by being radiated beneath the articular layers of both the MCC and the GF.23–25
The role of the MCC in the process of growth and development of the TMJ and its adaptive response to mandibular advancement are remarkably greater, particularly during the period of the present study.
This lower amount of activity in the GF might show that adaptation of the whole TMJ occurs mainly by ossification and relocation of the MCC and relocation of the GF, as a harmonized biologic response to mandibular protrusion. Otherwise, it is possible that the GF is not significantly relocated from its initial position, which could be a reason for future relapses of successful functional mandibular treatment. If the GF does not remarkably remodel or relocate, then the soft tissue attachments pull the condyle back to its initial relationship with the GF. For clearer and more detailed information in this regard, these possibilities should be precisely studied and evaluated by a combination of histochemical, cephalometric, and electromyographic methods over a longer period of time.
Signaling molecules of the FGF family regulate endochondral ossification at several levels26; therefore results of the current study are consistent with the fact that the mandibular condyle is a growth site and is ossified through endochondral ossification.22 However, endochondral ossification in the GF is observed only during initial stages of rats' growth (27 days of rats' age), as indicated by detection of FGF8 at that age and not after that age. This could indicate that osteogenesis generally slows down in rats' GF at this age, or that intramembranous ossification is the dominant ossification process in the GF afterward, and no cartilage tissue is found in the GF during later growth and development. Intramembranous ossification in the GF is similarly reported in other studies.27
Immunopositive cells for FGF8 molecules are detected in the cytoplasm of chondrogenic cells in the deep columnar proliferative layer and in early hypertrophic and hypertrophic zones.11,26 The location of FGF8 expression is consistent with other reports, which suggests that FGF8 could be known as an indicator for osteogenesis through an endochondral ossification process by patterning and regulating chondrocytes' proliferation and their hypertrophic morphologic differentiation.11,28,29
This indicates that FGF8 is more involved in hypertrophic activities than in chondrocyte proliferation. FGF8 plays a role in cellular chondrogenic differentiation and in creating morphologic changes from mesenchymal cells to chondroblasts and from chondroblasts to bone-making cells. This has also been reported by Minina et al.28 On the molecular level, FGF signaling reduces chondrocyte proliferation and induces hypertrophic differentiation of chondrocytes.28
The higher amount of FGF8 expression in experimental samples, in comparison with control samples, generally shows enhanced osteogenic transition occurring in both the MCC and the GF, with the exception of FGF8 excretion in the MCC on experiment day 30, during which the level of expression in control animals was slightly, although not significantly, higher than in the experimental rats. Additional long-term studies are required to find out the reason for the lower molecular and cellular activities in the GF than in the MCC.
Evaluating the expression of FGF8 in experimental rats versus controls suggests that mandibular advancement does not change the pattern of molecular activity, but just increases the level of activity. The pattern of FGF8 expression follows the pattern of normal growth and development of the TMJ, in reference to controls.
The FGF8 pick of expression is at approximately the same level in experimental and control rats. Considering this evidence and the fact that FGF8 in the condyle is slightly higher in controls on day 30, it is possible that stepwise advancement may generate more changes in the MCC-GF complex than one-step advancement. This is similarly suggested by other researchers.16,17
The future direction would be to design and perform studies covering longer periods of the rats' lives and comparing the MCC and the GF with other growth centers and growth sites on each sample, such as epiphyseal plates, synchondroses (eg, spheno-occipital synchondrosis), maxillary sutures (eg, intermaxillary suture), cranial sutures, and mandibular symphyses at earlier stages of growth. This could be done with cephalometric evaluations, in addition to histochemical evaluations, performed during the period of the experiment to measure the level and direction of growth and development.
CONCLUSION
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Structural and molecular adaptations occurred in the MCC and the GF of the experimental animals. Hypertrophic differentiations were significantly increased in both parts, which could enhance bone formation during the adaptive response. Therefore, mandibular growth modification takes place as an end result of extracellular morphologic differentiations and hypertrophic changes in the both the MCC and the GF.
Bite jumping appliance. Normal incisal relationship in control rats. The bite jumping appliance is adjusted on the lower incisors of experimental rats to move their mandible into a forward position during rest and function.
FGF8 in the MCC and the GF. Photograph of a female Sprague-Dawley rat's TMJ (A) shows the anatomic relationship of the condyle (A-MCC) and the articular fossa (A-GF), and the arrow (A) shows the direction of forward-downward displacement of the condyle during mandibular advancement. Photomicrographs show immunostaining for FGF8 expressed in the glenoid fossa of another experimental sample (27-day-old rat, wearing bite jumping appliance for 3 days) (B, C, and D) and the mandibular condylar cartilage of an experimental sample (38-day-old rat, wearing bite jumping appliance for 14 days) (E, F, and G).
FGF8c and FGFgf: diagram for experimental rats vs controls on different experiment days. In this diagram, the means of the numbers of FGF8-immunopositive cells in the mandibular condylar cartilage (FGF8c) and in the glenoid fossa (FGF8gf) are compared in experimental and control rats on different experiment days.
Contributor Notes