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Original article
Unilateral nasal obstruction induces morphologicalchanges of the mandibular condylein growing rats
Kenzo Watakabe, Ikuo Yonemitsu *, Yuhei Ikeda, Tang Huan,Takashi OnoDepartment of Orthodontic Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and DentalUniversity, Tokyo, Japan
a b s t r a c t
Purpose: Chronic nasal obstruction is known to decrease blood oxygen saturation. Mouth
breathing in association with chronic nasal obstruction leads to the collapse of the
buccinator mechanism and to a clockwise rotation of the mandible, which causes
mandibular retrusion. This study aimed to investigate the influences of nasal
obstruction on the morphological and histological changes of the mandible in growing
rats.
Materials and methods: Thirty 8-day-old male Wistar rats were randomly divided into the control
and experimental groups. The experimental group underwent unilateral nasal obstruction by
cauterization of the external nostrils at 8days of age. Pulse oxygen saturation (SpO2) was
monitored every week. Rats were sacrificed at 9 weeks of age. The mandibular changes were
analyzed via lateral cephalometric radiographs and micro-CT scans. We utilized toluidine blue
and tartrate-resistant acid phosphatase (TRAP) staining for histological analysis. Immunohisto-
chemical staining of hypoxia induced factor-1a (HIF-1a), vascular endothelial growth factor
(VEGF), osteoprotegerin (OPG) receptor activator of nuclear factor kappa-B ligand (RANKL) were
also performed to reveal the mechanism of the morphological changes.
Results: SpO2 was significantly lower in the experimental than in the control group. In the
experimental group, length, bone mineral density and cartilage layer thickness of
mandibular condyle were decreased. The number of TRAP-positive cells in the condyle,
HIF-1a-positive cells, VEGF-positive cells and RANKL-positive cells in the condylar
cartilage was significantly increased. In contrast, a reduced expression of OPG protein was
observed in the experimental group.
Conclusions: Our findings suggest that unilateral nasal obstruction in the growth period
affects mandibular morphology.
© 2018 Elsevier Ltd and The Japanese Orthodontic Society. All rights reserved.
a r t i c l e i n f o
Article history:
Received 17 October 2017
Received in revised form
18 May 2018
Accepted 22 May 2018
Available online 11 June 2018
Keywords:
Condyle
Hypoxia
Hypoxia-inducible factor-1a
Micro-CT
Nasal obstruction
* Corresponding author at: Department of Orthodontic Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and DentalUniversity, 1-5-45 Yushima Bunkyo-ku, Tokyo 113-8549, Japan.
E-mail addresses: [email protected] (K. Watakabe), [email protected] (I. Yonemitsu), [email protected] (Y. Ikeda),[email protected] (T. Huan), [email protected] (T. Ono).https://doi.org/10.1016/j.odw.2018.05.0011344-0241/© 2018 Elsevier Ltd and The Japanese Orthodontic Society. All rights reserved.
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1. Introduction
Recently, the prevalence of allergic rhinitis has increasedworldwide, occurring in 10% to 30% of adults and up to 45% ofchildren [1]. Allergic rhinitis is a representative symptom of nasalrespiratory disorder characterized by three major symptoms:sneezing, nasal mucus secretion, and nasal obstruction (NO).Chronic NO is known to decrease pulse oxygen saturation (SpO2)[2]. Further, lung weight reduction and hormonal behavioralchanges have been reported in rat models of bilateral NO [3,4].
Previous studies have demonstrated how mouth breathingassociated with rhinostenosis influences the function of thebuccinators. Mouth breathing induces lip dysraphism andtriggers the clockwise rotation of the mandible further causingmandibular retrusion and Class II malocclusion in humans [5].Recent studies have revealed a relationship between NO andmalocclusion. Mouth breathing combined with nasal conges-tion induces a vascular mechanism collapse that leads toadenoid facies resulting from the backward rotation of themandible [6]. This combination has also been found to causeinterference of the lip closure, high palate, and Angle Class IImalocclusion in humans [7]. Unilateral NO (UNO) was found tosuppress the jaw-opening reflex in a rat model [8]. In addition,it has been reported that NO affects not only muscle reflexesbut also the growth of both muscle and bone in the rat model[3,9]. In the craniofacial area, decreased growth of the massetersuperficial layer and the anterior belly of the digastric muscle,including the vertical reduction of the nasomaxillary complex,were observed in a rat model of bilateral NO [10,11]. Insummary, NO has various effects on the craniofacial region;however, the underlying mechanisms of the mandibularmorphological changes associated with NO are still unclear.
Hypoxia induced factor-1 alpha (HIF-1a) expression isassociated with the decrease of SpO2 under hypoxic conditionin rabbits [12]. It is activated during hypoxia and involved inosteogenesis and angiogenesis [13]. It was also reported thatHIF-1a acts on the vascular endothelial growth factor (VEGF) topromote osteoblastic and osteoclastic differentiation andregulate chondrocyte apoptosis through the glycolysis systemin rats [14,15]. In addition, the osteoclast activation mechanismof HIF-1a has previously been reported: hypoxia upregulatesthe glycolysis pathway and increases the expression of glucosetransporters [16]. Subsequently, HIF-1a expression increases inosteoclasts with the release of cathepsin K and hydrogen ionsresulting in bone resorption in mice [17].
Osteoprotegerin (OPG) is another protein that is reportedlyrelatedtoosteoclastactivation.And,OPGisdownregulatedunderhypoxia, thus promoting osteoclast activation in rats [18]. OPGacts as the receptor activator of nuclear factor kappa-B ligand(RANKL) decoy receptor in the RANK-RANKL system. Generally,itsupregulationsuppressesosteoclastactivitythroughbindingtoRANKL in vitro [19]. An increased RANKL/OPG ratio and adecreased number of OPG-positive cells were detected in theprevious study using osteoarthritic TMJ of rats [20].
However, to our knowledge, no study has yet investigatedthe relation between the chronic hypoxia caused by the NO-related expression of HIF-1a, OPG, RANKL and tartrate-resistant acid phosphatase (TRAP) activity and the reductionin bone volume. In this study, we evaluated the influence of
UNO on the mandibular morphology of growing rats throughthe assessment of the HIF-1a expression, OPG expression,RANKL expression and TRAP activity in the condyle.
2. Materials and methods
2.1. Animal preparation
Animal protocols were approvedbythe Institutional AnimalCareand Use Committee of the Tokyo Medical and Dental University(#0170370A), and the experimental procedures were performedin accordance with the University’s Animal Care Standards.
Thirty 8-day-old male Wistar rats were used in thisexperiment. The rats were randomly divided into the controland experimental groups (n=15 each). All rats were firstanesthetized by hypothermia (10min at �18�C). UNO wasinduced in the experimental group by cauterization of theexternal nostril on post-natal day 8. Cauterization wasperformed by burning the surrounding tissues of the leftnostril, using a 400�C-stainless-steel wire that was 1mm indiameter. For the first week, we visually checked every daywhether cauterized noses remained closed; we re-cauterizedeach nose immediately if it re-opened. After 1 week, weobserved cauterized noses once per week and verified thatthey never opened during the experimental period. Thecontrol group underwent a sham operation in which thecauterizing instrument was placed 1–2mm above the leftnostril. 3% chlortetracycline (Aureomycin
1
Ointment; PolaPharma, Tokyo, Japan) was applied on the left external nostrilof rats in both groups postoperatively to prevent infection [21].Weight and pulse oxygen saturation were monitored everyweek using a pulse oximeter (MouseOx
1
STARR Life SciencesCorp., Oakmont, PA). Both the experimental and control groupanimals were euthanized at 9 weeks using CO2 gas.
2.2. Radiological analysis of the mandible and tibia
The radiological analysis was performed using the proceduresreported previously [22]. Briefly, lateral radiographs were ob-tained with a soft X-ray system (SOFTEX CMB-2; SOFTEX Co., Ltd.,Tokyo, Japan) to evaluate craniofacial morphological changes.The head of each rat (n=15 each) was fixed using a pair of ear rodsto maintain a standard head position. The head was maintainedin contact with the film to reduce the magnification factor [23].ThevariouspartsofthemandibularboneweremeasuredwiththeNIHimagesoftware(NIS-Elements Analysis D,National Institutesof Health, Bethesda, MD, USA). The measurement points areshown in Table 1 and Fig. 1. Selected linear measurements werethen obtained (Table 2). The cephalometric landmarks (Table 1,Fig. 1) were derived from the previous studies in rodents [24,25].The soft X-ray settings were 50kVp, 15mA, and 5-s impulse [23].The whole tibia was collected and its length was measured as anindicator of whole body growth. All radiographs were taken threetimes by the same operator.
2.3. Microcomputed tomography analysis (Micro-CT)
Micro-CT analysis and a desktop X-ray micro-CT system (SMX-100CT; Shimadzu, Kyoto, Japan) were utilized to investigate the
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changes in the bony microstructure of mandibular condyles andtibias of rats (n=15 each). The region of interest (ROI;1.0�1.0�0.2mm) was defined as the region 1.0-mm distantfrom the articular cartilage of the mandibular condyle, todistinguish the ROI from the cortical bone area. Each mandibularcondyle was analyzed using three-dimensional image-analysis
software (TRI/3D-BON; Ratoc System Engineering, Tokyo, Japan).A scanning resolution of 20mm, which is recommended for theanalysis of bone density, was used to assess the mandibularcondyle in the cancellous bone of the mandibular condyle [15](Fig. 2). The tibia lengths were also measured using the samemethods as for condyle measurement.
2.4. Tissue preparation
The temporo-mandibular joints (TMJs) of both sides, alongwith their surrounding tissues were immersed in 4% bufferedparaformaldehyde at 4�C for 24h and decalcified with 4% EDTAfor 8 weeks at 4�C. Then, they were embedded in paraffin, andserial sections of 6.0-mm thickness were cut through thesagittal plane [26].
2.5. Histomorphometry with toluidine blue staining
The sagittal sections of the center of the condyle were stainedwith toluidine blue to measure the width of the mandibularcondylar cartilage layers and to observe the chondrocytes. Fromthe articular surface down, the condyle was divided into thefibrous, proliferative, mature, and hypertrophic layers. Thecondylar cartilage was divided into three areas; the anterior,posterior, and superior regions. The anterior region consisted ofthe anterior half of the cartilage, the posterior region consistedof the posterior quarter of the cartilage, and the superior regionconsisted of the area between these two regions [27]. Measure-ment of the width of thecartilage layer of the anterior, posterior,and superior regions was carried out using image analysissoftware (NIS-Elements Analysis D, National Institutes ofHealth, USA) (n=15 each) [28].
2.6. Enzymatic staining of tartrate-resistant acidphosphatase (TRAP) activity
Histochemical analysis of TRAP activity in the mandibularcondylar bone was performed to detect osteoclasts using theprepared sections. A positive reaction to TRAP activity is aknown marker of mature osteoclasts. TRAP staining wasperformed using a TRAP/ALP Stain Kit (Wako; Tokyo, Japan)(n=15 each) [29]. Quantitative analysis was performed imme-diately below the hypertrophic layer in the superior region ofthe condylar cartilage.
Fig. 2 – The region of interest (ROI) was located in the cancellous bone of the mandibular condyle (white box). Abbreviations: M,mesial; D, distal. Bar indicates 500mm.
Fig. 1 – The landmarks used for the measurements on the softX-ray images of the rats.
Table 2 – Linear measurements of the mandible.
Go-Mn Posterior corpus lengthM1-L1 Anterior corpus lengthCo-L1 Total mandibular lengthCo-Gn Ramus heightCo-Mn Ramus length
Table 1 – Definitions of landmarks.
Co The most posterior and superior point on the mandibularcondyle
Go The most posterior point on the mandibular ramusMn The most concave portion of the concavity on the inferior
border of the mandibular corpusGn The most inferior point on the ramus that lies on a
perpendicular bisector of the line Go-MnL1 The most anterior and superior point on the alveolar bone
of the mandibular incisorM1 The junction of the alveolar bone and the mesial surface of
the first mandibular molar
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2.7. Anti-HIF-1a antibody and anti-OPG staining
We evaluated HIF-1a, VEGF, OPG and RANKL proteinexpression in the mandibular condylar cartilage of eachrat (n=15 each) using the prepared sections at the same siteas the toluidine staining and TRAP staining. Monoclonalmouse anti-HIF-1a (BD Pharmingen, San Diego, CA, USA),monoclonal mouse anti-VEGF (LVC, MS-350-P0, CA, USA),monoclonal goat anti-OPG (sc-8468, Santa Cruz Biotechnolo-gy, CA, USA), and polyclonal rabbit anti-RANKL (sc-7628,Santa Cruz Biotechnology, CA, USA) antibodies were used asprimary antibodies, at a dilution of 1:100, 1:100, 1:50, and1:50, respectively. Simple Stain MAX-PO (NICHIREI BIO-SCIENCES, Tokyo, Japan) was used as secondary antibodies.In each section, the number of HIF-1a, VEGF, OPG andRANKL-positive cells were counted using a fixed measuringframe (450 mm�900mm) at least three times. The measuringframe covered the total thickness of the proliferative andhypertrophic layers in the superior region of the mandibularcondyles as previously reported [29,30]. In addition, the
RANKL / OPG ratio was calculated in order to reveal changesin osteoclastogenesis, as previously reported [31].
2.8. Statistical analysis
After testing for normality and equal variances, the Student’st-test was used to compare mean values among groups. Allstatistical tests were performed using SPSS ver. 2.0 (SPSS JapanInc., Tokyo, Japan). A P-value <0.05 was considered significant.
3. Results
3.1. Systemic changes in rats
The body weight, tibial length, and cancellous bone volumedensity in the tibia of rats were measured as an indicator indexof whole body growth. There was no significant differencebetween the control and experimental groups (Fig. 3A).However, the value of SpO2 in the experimental group was
Fig. 3 – (A) (i) Changes in body weight of rats during the experimental period. Values are depicted as the mean�standarddeviation (S.D.). There were no significant differences between the two groups at any stage (n=15 each). (ii) The length of thetibias of 9-week-old rats. Values are mean�S.D. There were no significant differences between the two groups (n=15 each). (iii)Comparisons of bone morphology between the control and experimental group by micro-CT analysis. The cancellous bone ofthe tibia was compared between the control and experimental groups (n=15 each). (B) Changes in pulse oxygen saturation(SpO2) of rats during the experimental period (n=15 each). Values are depicted as the mean�S.D. There were significantdifferences between the two groups from 2 to 5, and 7 to 9 weeks of age. *: p<0.05. Abbreviation: BV/TV, bone volume/tissuevolume.
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significantly decreased from 2 to 5, and 7 to 9 weeks of agecompared to that in the control group (Fig. 3B).
3.2. Cephalometric measurements
Intheexperimental group, Go-Mn andCo-Mn were significantlyshorter than in the controls. These changes indicate that thelength of the mandibular ramus was shorter in the experimen-tal compared with the controlgroup(Fig. 4). However, the lengthof the M1-L1, Co-L1, and Co-Gnshowed no significant differencebetween the control and experimental groups.
3.3. Three-dimensional micro-CT analysis
The experimental group showed a lower cancellous bonevolume density compared with the control group. The micro-CT analysis demonstrated significantly lower BMD and BV/TVof the mandibular condyle in the experimental groupcompared to the control group (Fig. 5).
3.4. Histomorphometry with toluidine blue staining
Chondroblastic layers consisted of four layers from thesuperior of the condyle: fibrous, proliferative, mature, andhypertrophic layers (Fig. 6A). The layers in the superior regionof the experimental group were thinner than those in the
control group. The cartilage in the anterior, posterior, andsuperior region in the experimental group was significantlythinner than at the corresponding regions in the control group(Fig. 6B). Metachromatic staining was observed in thetransitional and hypertrophic layers of the condylar cartilageof both groups. From the mature to the hypertrophic layers, thewidth of the cartilage in the experimental group changedthinner than in the control group.
3.5. Quantitative analyses of osteoclastic cell numbers
The TRAP-positive cells immediately below the hypertrophiclayer of the condyles were observed in both the experimentaland control groups (Fig. 7A). The number of TRAP-positive cellsin the superior region of the condyle was significantly higher inthe experimental group (Fig. 7B).
3.6. Expression of HIF-1a, VEGF, OPG and RANKL proteinin the articular cartilage of the rat mandibular condyle
Immunohistochemical analyses showed that the HIF-1a(Fig. 8A) and VEGF (Fig. 8C) protein were expressed specificallyin the mature hypertrophic cell layer of the mandibularcondylar cartilage of the experimental group. The ratio of HIF-1a- (Fig. 8B) and VEGF- (Fig. 8D) positive cells were significantlyhigher in the experimental group than in the control group.
Immunohistochemical analyses showed that OPG proteinexpression was increased in the mandibular condyle of thecontrol group. In contrast, OPG was barely detected in theexperimental group (Fig. 9A). The ratio of OPG-positive cellswas significantly higher in the control group compared to theexperimental group (Fig. 9B). However, there was no signifi-cant difference in RANKL protein expression between controland experimental groups (Fig. 9C). The RANKL/OPG ratio wassignificantly higher in the experimental group, compared withthe control group (Fig. 9D).
4. Discussion
It has been reported that bilateral nasal obstruction in ratsgreatly affects the growth and development of the whole body[3,9]. However, in our study, when the BMD and length of thetibia were measured as whole body indices, no significant
Fig. 5 – (A) Comparisons of bone morphology between the control group and experimental group by micro-CT analysis. Thecancellous bone of the mandibular condyle was compared between the control and experimental groups. Abbreviations: BMD,bone mineral density; BV/TV, bone volume/tissue volume. Data were shown as medians with standards errors for each group *:p<0.05. (n=15 each).
Fig. 4 – The results of morphological analysis of themandibular bone. The length of the Go-Mn and Co-Mn weresignificantly decreased. Therefore, the length of the mandib-ular ramus was shortened. *: p<0.05. (n=15 each)..
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differences were observed, suggesting that UNO has littleeffect on whole body growth. As we used a unilateral nasalobstruction model rather than a bilateral nasal obstructionmodel, we had to determine whether unilateral change hadoccurred. As a result, no significant difference was observedbetween left and right sides.
Even though there was little effect on the other parts of thebody, the UNO animals showed a shortened mandibularramus, decreased BMD of the mandibular condylar cancellousbone, and thinner condylar cartilage. In the UNO group, thevalue of SpO2 was significantly decreased from 2 to 5 and 7 to9 weeks of age, compared to the control group. The value ofSpO2 stabilized at about 93% after 7 weeks of age. These resultsare almost in agreement with those of previous studiesinvolving rats [32,33]. Nasal obstruction is related to an initialdecrease in lung growth [3], and rat lung growth is reported tooccur at 3 weeks of age [34,35], while capillary growth, whichplays an important role in optimizing gas exchange, occurs at2–5 weeks of age [34,36]. In our experiments, we assumed thatthe experimental period influenced lung and capillary growth.The significant decrease in SpO2 from 6 weeks of age in theexperimental group was likely due to a reduction in ventilationafter the completion of the growth of lungs and capillaries, as isseen in humans with nasal obstruction [37].
In the radiological analysis of the mandible, the posteriorcorpus length and ramus length were significantly shorter in theexperimental group. However, the anterior corpus length, totalmandibular length, and ramus height were not significantlydecreased in the experimental group. According to the previousstudy, nasal obstructionis associatedwithupward andbackwardgrowth of the condyle, and a decreased vertical component ofgrowth in monkeys [38]. Additionally, the directional growth ofthe rat mandible is affected by orofacial functional changes [9].
Our findings and those of other groups have revealedvarious possible mechanisms for the morphological changesof the mandible caused by NO. It was reported that themandible rotates clockwise due to the nasal obstruction andassumes a mandibular posture similar to open bite. Thiscauses decreased growth of the mandibular ramus, similar tothe results of the current study. Furthermore, it was reportedthat in the case of such a mandibular posture, the activity ofthe jaw-closing muscle of the mandible reduces and theweight of the masseter muscle decreases [38]. In line with this,we observed a decreased length of the mandibular ramus inour experimental group, which implies a lower massetermuscle mass and less mandibular ramus growth.
According to another previous study, body growth wasdelayed in a short-term unilateral nasal obstruction model after
Fig. 6 – (A) Histological staining with toluidine blue of 6-mm sagittal sections of 9-week-old rat condyle: (i) control and (ii)experimental groups (n=15 each). Metachromatic staining was observed in the transitional and hypertrophic layers of thecondylar cartilage of both groups. Bar indicates 100mm. Abbreviations: F, fibrous layer; P, proliferative layer; M, mature layer; H,hypertrophic layer. (B) The width of the chondroblastic layers of the superior region of 9-week-old rat condyles was measured.Values are depicted as the mean�S.D. *: p<0.05.
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birth; and low levels of glucocorticoids caused the decreasedgrowth of the rat craniofacial area. However, at 90-days-old,there were no significant differences in body weight [9].Considering the results of this previous study, it is possiblethat the level of growth hormone was also decreased in ourmodel, which may have resulted in reduced growth of themandible.
It is widely known that the mandibular condylar growth iscaused by endochondral ossification; however, there havebeen no studies focused on the relationship between expres-sion of HIF-1a and endochondral ossification in a nasalobstruction model. It has been reported that HIF-1a is involvedin the apoptosis of chondrocytes [42] and is related to adecrease of cartilage thickness in the rat mandibular condyle[29], which is concordant with our findings.
It is possible that the whole body decrease in SpO2 arisingfrom the nasal obstruction caused a localized reduction ofSpO2 at the TMJ, thereby affecting the mandibular condylarcartilage. A previous study reported that SpO2 in the synovialfluid of rheumatoid arthritis patients is lower than in that ofhealthy subjects [43]. Similarly, the reduction of SpO2 in themandibular condylar cartilage might have also occurred innasal obstruction model [38–41]. Although HIF-1a promotescell survival under normal circumstances [44], its expression
under hypoxic conditions can activate pathways that lead tosynovitis, angiogenesis, cartilage degradation, and boneerosion [45–48]. It was reported that the expression of HIF-1aincreases in the mandibular condylar cartilage of rats underhypoxia [49]. Our results also suggested that the increasedexpression of HIF-1a in the mandibular condylar cartilage, dueto systemic hypoxia with nasal obstruction, may have causedcartilage degradation and reduction of bone density on themandibular condyle.
In mandibular bone morphometry, a decrease in mandibu-lar ramus length and in mandibular bone density wereobserved, but no change was observed in the tibia. This issimilar to that in the intermittent hypoxia (IH) model involvingrats [50]. Breathing interacts with chewing and swallowing,etc., thereby allowing proper growth and development of thecraniofacial area. Chronic nasal respiratory disturbance isoften observed in children's nonspecific respiratory deformi-ties such as adenoid hypertrophy [9]. The difference betweenthe results in the mandibular bone and the tibia may be due tothe fact that the nasal obstruction causes physiological andfunctional changes in the mandibular anterior muscles,resulting in a change of muscle function in the craniofacialarea. This thus leads to a large influence on the mandibularbone. We are currently studying the degeneration of the
Fig. 7 – (A) Tartrate-resistant acid phosphatase (TRAP) staining in the superior region of 9-week-old rat mandibular condyle. (i)Control and (ii) experimental groups. TRAP-positive cells were seen in osteoclasts beneath the cartilage layer. Bar indicates100mm. (B) The number of TRAP-positive cells found in 9-week old rat condyles (n=15 each). Quantitative analysis was carriedout immediately below the hypertrophic layer in the superior region of the condylar cartilage. Values are depicted as the mean�S.D. *: p<0.05.
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Fig. 8 – (A) Representative immunohistochemical staining with anti-HIF-1a antibody in both the (i) control and (ii) experimentalgroups. Arrow heads indicate HIF-1a-positive cells. Bar indicates 100mm. (B) HIF-1a-positive cells were counted in each group(n=15 each) using a fixed measuring frame (450mm�900mm). The percentage of HIF-1a positive cells was calculated as thenumber of positive cells to the total number of counted cells. Values are depicted as the mean�S.D. *: p<0.05. (C) Expression ofVEGF in the condylar cartilage of rat temporo-mandibular joints. Representative immunohistochemical staining with anti-VEGF antibody in both the (i) control and (ii) experimental groups. Bar indicates 100mm. (D) The percentage of VEGF-positivecells was calculated as the number of positive cells per total number of cells (n=15 each). Values are depicted as the mean�S.D. *:p<0.05.
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Fig. 9 – (A) Expression of OPG in the condylar cartilage of rat temporo-mandibular junctions. Representative immunohisto-chemical staining with anti-OPG antibody in both the (i) control and (ii) experimental groups. Bar indicates 100mm. (B) Thepercentage of OPG-positive cells was calculated as the number of positive cells per total number of cells (n=15 each). Values aredepicted as the mean�S.D. *: p<0.05. (C) Expression of RANKL in the condylar cartilage of rat temporo-mandibular joints (n=15each). Representative immunohistochemical staining with anti-RANKL antibody in both the (i) control and (ii) experimentalgroups. Bar indicates 100mm. (D) The ratio of RANKL/OPG was calculated (n=15 each). Values are depicted as the mean�S.D. *:p<0.05.
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masticatory muscle in a specific 9-week-old nasal obstructionmodel.
Several experimental hypoxic stress models have demon-strated the effects of IH for rats within the hypoxic chambers[51]. In particular, IH has been shown to lead to a decreasedmandibular ramus height and increased mandibular bonedensity [24]. In the IH model, oxygen saturation is lower thanthat in normal subjects, similar to the reduced SpO2 due toUNO in the current study. This might be the reason for thesimilarity in findings. In both models, no significant differencewas observed in the tibial length; significant differences wereonly observed in the craniofacial area bones. However, withregards to the BMD, the results of our study differed: the BMD ofthe mandibular condylar cancellous bone was decreased in theUNO group. This could be the consequence of differences in thebreeding age of rats and the oxygen saturation levels. In ourmodel, the rat's breeding age was 8days old to 9weeks old andthe oxygen saturation in the UNO was chronically about 94%,whereas in the IH model, the breeding age was from 7 to10 weeks old and the oxygen saturation intermittently rangedfrom 4% or 5% to 21%. [50,52].
There are various opinions regarding the effects of hypoxiaon BMD. A previous study claimed that chronic IH does notinfluence BMD [53]. In contrast with the previous studies, wefocused on the period from early childhood until the end of thegrowth. Therefore, the decrease of BMD observed in this studymight be related to the difference in the age of rats compared toprevious experimental models. Moreover, we found that theexperimental group had a chronic decrease in SpO2 from 8daysto 9weeks of age. Because decrease in SpO2 was chronic, andnot intermittent or long-term, our results were different fromthose of the IH model. Based on the above, we believe that thechanges in bone mineral density were due to multiple factors,including changes in masticatory muscle activity, age, andhypoxic exposure time.
HIF-1a is one of the important markers for evaluating BMDand chondrocyte layer thickness in the NO model because itrelates to the hypoxic stress condition, osteoclast differentia-tion, and chondrocyte apoptosis [42]. In the UNO group, thenumber of HIF-1a-positive cells increased and the chondro-cyte thickness decreased significantly, which is similar to theprevious findings regarding HIF-1a expression [29,54]. Simi-larly, a correlation exists between the expression of TRAP-positive cells and HIF-1a-positive cells [55]. We observed asimilar increase in the number of both HIF-1a-positive andTRAP-positive cells in the UNO group. The TRAP positive-cellsof secondary spongiosa, corresponding to the ROI of micro-CTin the UNO group, were also increased; moreover, osteocytesand bone marrow were disarranged in the secondary spon-giosa in the UNO group. These were consistent with the resultsof micro-CT. As for OPG and RANKL, we observed theactivation of osteoclasts and reduction of OPG, the increasedRANKL/OPG ratio was similar to previous reports [19,56,57].Therefore, we suggest that the expression of HIF-1a decreasesthe expression of OPG, leads to an increased RANKL/OPG ratio,resulting in an increase of the number of TRAP-positive cellsand a reduction of bone density. Moreover, we found adecrease of the chondrocyte layer thickness in the experi-mental group, confirming a previous finding that HIF-1aexpression promotes chondrocyte apoptosis [58]. In addition,
the aforementioned mechanism is also conceivable due to thefact that HIF-1a and OPG are expressed in the mature andhypertrophic layers, and that the number of osteoclastsimmediately under the hypertrophic layer is increased.Furthermore, the toluidine blue staining in the experimentalgroup showed enlarged and roughened chondrocytes. Thisappearance was more frequently observed in the hypertrophicchondrocyte layer. This is similar to the observations ofchondrocytes during apoptosis in a previous study [59].
5. Conclusions
This is the first study to report the histological effects of nasalobstruction on the mandible. Our results indicate that nasalobstruction during the growth period causes morphologicaland histological changes in the mandible.
Conflicts of interest
None to declare.
Funding
This study was supported in part by Grants-in-Aid forScientific Research (16K11782) from the Ministry of Education,Culture, Sports, Science and Technology of Japan.
Ethical approval
The experimental procedures described here were approvedby the Institutional Animal Care and Use Committee(#0170370A) and performed in accordance with the AnimalCare Standards of Tokyo Medical and Dental University.
Acknowledgements
This study was financially supported by Grants-in-Aid forScientific Research (16K11782) from the Ministry of Education,Culture, Sports, Science and Technology of Japan.
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