10.59987/ads/2017.3.95-103
Mandible condyle remodeling is a great challenge on craniofacial growth studies. The great majority of the reports deals with growing period. However, there is a great necessity to clarify the importance of functional stimulation on adult mandible condyle remodeling. By using an adult mouse model, we investigated the influence of mandible forwarding on condyle remodeling and gene expression by bone forming cells. Tomographic and scintigraphic evaluations showed sagittal growth and cell activity enhancement. RT-PCR showed that Type I collagen, osteocalcin and osteonectin expression level can be altered. We showed that functional stimulation is necessary to maintain the regular gene expression by condyle bone forming cells in adult mice. It opens new frame for further investigations aiming new clinical approaches to temporomandibular joint problems treatment, as well as mandible retrusion treatment.
application/pdf
1971-1441
51
3
en
F. Leite 
https://creativecommons.org/licenses/by-nc-nd/4.0
Annali di stomatologia
gene expression
Mandible protraction alters Type I collagen, osteocalcin and osteonectin gene expression in adult mice condyle
Text.Serial.Journal
https://www.annalidistomatologia.eu/ads
8
Original Article
2022-08-10
2022-08-10
2017-09-01
2023-12-04
95-103
Ann Stomatol (Roma)
F. Leite 
2017
10.59987/ads/2017.3.95-103
95
1971-1441
3
gene expression
en
103
Mandible protraction alters Type I collagen, osteocalcin and osteonectin gene expression in adult mice condyle
https://www.annalidistomatologia.eu/ads/article/download/51/39
8
                                                                                                Original article


    Mandible protraction alters Type I collagen,
    osteocalcin and osteonectin gene expression
    in adult mice condyle




                                                                                             li
                                                                                           na
    Patricia Valerio1 PhD                                  Introduction

                                                           Head and face growth disorders are very common in
    Filipi Perfeito2 DDS

                                                           all racial populations. The prevalence of craniofacial
    Livia P. Moura1 MDS




                                                                                         io
                                                           anomalies varies among different ethnicities based
    Deborah N. Ribeiro1 MDS

                                                           on genetic background, geography, socio-economic
    Simone O. A. Fernandes2 PhD
    Almir S. Martins1 PhD
                                                           status and environmental factors (1). Mandible retru-




                                                                     az
    Maria F. Leite1 PhD
                                                           sion prevalence runs from 52 to 56% (2), and the
                                                           most common treatment is condyle traction generat-
                                                           ed by the mandible forward repositioning. Mandible
    1 Department of Physiology and Biophysics,             protrusion enhances functional stimulation on condyle
    Federal University of Minas Gerais, Brazil




                                                                   rn
                                                           and generates sagittal growth et al. (3). However, we
    2 School of Pharmacy, Federal University of
                                                           have a huge controversy. Some reports indicate a
    Minas Gerais, Brazil                                   positive growth response to condyle traction while
                                                           others indicate a negative or no response to




                                                    te
                                                           mandibular advancement (4). Therefore, there is a
                                                           need to identify the markers for each stage of condy-
                                                           lar growth and evaluate the effect of functional stimu-
    Patricia Valerio
    Corresponding author:
                                                  In
                                                           lation on growth pattern. Condyle grows by endo-
    Department of Physiology and Biophysics,
                                                           chondral ossification, and some Authors have been
    Federal University of Minas Gerais
                                                           studying the early stages of condyle formation, focus-
    Cristiano Machado 1630/901
                                                           ing on the importance of functional stimulation for
    31170024 Belo Horizonte, Brazil
                                                           chondrogenesis. However, the osteogenic phase of
                               ni

    E-mail: patricia.valerio@terra.com.br
                                                           condyle formation has been missed. The possible
                                                           gene expression alteration on this phase, generated
                                                           by different pattern of stimulation, was not studied yet
                             io


                                                           (5-10). Moreover, the majority of the reports deals
                                                           with growing period and do not focus on adult phase
    Summary
                                                           (11-16). Considering not only mandible retrusion cor-
                           iz



                                                           rection but also considering that remodeling alteration
    Mandible condyle remodeling is a great challenge

                                                           is the main cause for articular temporomandibular
    on craniofacial growth studies. The great majority

                                                           disorders affecting many adults (17), it is important to
            Ed




    of the reports deals with growing period. Howev-

                                                           clarify the mechanisms involved on adult condyle re-
    er, there is a great necessity to clarify the impor-

                                                           sponse to functional stimulation. So, our aim was to
    tance of functional stimulation on adult mandible

                                                           evaluate the alteration on osteoblast gene expression
    condyle remodeling. By using an adult mouse

                                                           pattern, on mouse adult condyle, under different func-
    model, we investigated the influence of mandible

                                                           tional stimulation.
    forwarding on condyle remodeling and gene ex-
    pression by bone forming cells. Tomographic and
 IC




    scintigraphic evaluations showed sagittal growth
    and cell activity enhancement. RT-PCR showed
    that Type I collagen, osteocalcin and osteonectin      Materials and methods
    expression level can be altered. We showed that
C




                                                           This study was approved by the Federal University of
    functional stimulation is necessary to maintain        Mandible protrusion protocol

                                                           Minas Gerais Animal Use and Care Committee (Pro-
    the regular gene expression by condyle bone

                                                           tocol 169/2014). 48 female C57 mice (including 24 as
    forming cells in adult mice. It opens new frame for

                                                           controls) were used in this work. We decided to in-
    further investigations aiming new clinical ap-
©




                                                           vestigate the effect of mandible protrusion on condyle
    proaches to temporomandibular joint problems

                                                           osteoblasts gene expression. In order to generate
    treatment, as well as mandible retrusion treat-

                                                           mandible protrusion, 24 mice (experimental group)
    ment.

                                                           had the inferior incisors cut around 1 mm. We used a
                                                           nail plyer, holding the animal in left hand and using
    Key words: bone biology, growth evaluation, mol-

                                                           the plyer on right hand to cut the inferior incisors. It
    ecular biology, CT, condyle growth, gene expres-
    sion.

    Annali di Stomatologia 2017;VIII(3):95-103                                                                  95
    F. Perfeito et al.


    generates an increase on the overjet. Because of                  lets and tap water and were housed at the animal ex-
    this, they had the necessity of sending the mandible              perimentation laboratory. Every 3 days the cutting
    forward to be able to chew (Fig. 1).                              procedure was repeated, due to continuous eruption
    The weight of each animal was checked. Control                    of the teeth, as well as the weight checking.
    mice only underwent weight checking to evaluate the
    regular food intake and consequent weight gain. The
    animals were provided with free access to food pel-               To evaluate the condyle sagittal shape changing, we




                                                                                                            li
                                                                      Tomography




                                                                                                          na
     a




                                                                                                        io
                                                                                  az
                                                                                rn
                                                             te
                                                           In
                               ni

     b
                             io
                           iz
            Ed
 IC
C




    Figure 1 a, b. Mandibular advancement. (a) To generate a physiological mandibular advancement, that would lead to stimu-
©




    lation on condyle region, every 3 days during 21 days, the lower incisors were cut by 1 mm to induce protrusion when the
    animal was feeding. In left panel we can see the control animal with regular distance between inferior and superior incisor,
    and on right panel we can see the experimental animal immediately after cutting, showing an increase on the overjet; (b) the
    weight gaining of the animals indicated that on the first days the experimental group was not used to the increase of overjet
    and had weight loss, but after 9 days they started to eat normally and recover the weight gaining (first point of the graphic is
    3 and last point of the graphic is 18, because we checked the weight on the cutting day).


    96                                                                                     Annali di Stomatologia 2017;VIII(3):95-103
    Mandible protraction alters Type I collagen, osteocalcin and osteonectin gene expression in adult mice condyle


    used a tomographic study. The animals were anes-
    thetized with a combination of ketamine (30 mg/kg)               The 99mTechnetium-methylene diphosphonate (99mTc-
                                                                     Scintigraphy

    and xylazine HCl (2.5mg/kg) intramuscularly. Then,               MDP), a specific marker of the anabolic phase of re-
    they were positioned on a table adapted to the equip-            modeling, has been used in the diagnosis of a broad
    ment as showed on the Figure 2a. The condyle was                 spectrum of conditions affecting the skeleton. The ra-
    localized on frontal and coronal plane and measure-              diopharmaceutical 99m Tc-MDP was obtained from
    ments were done on sagittal plane (Fig. 2b). We used             UFMG Pharmacy school laboratory. To observe bone




                                                                                                          li
    a Gendex CB500 with voxel 0.125 and 4 cm high.                   metabolic activity in the region of the condyle, bone




                                                                                                        na
                                                                                                      io
                                                                                az
                                                                              rn
                                                            te
                                                          In
      a                                                                 c
                               ni
                             io
                           iz
            Ed
 IC
C




     b
©




    Figure 2 a-c. Tomography. (a) After anesthesia, the animals were positioned on the equipment table as showed on the pic-
    ture; (b) The software used allows the user to measure linear distances in 3 dimensions. After anesthesia, the animals were
    analyzed in coronal (right panel) and transversal (left upper panel) plane to localize the condyle. The measurement was
    done on sagittal plane (left lower panel). Measurements were done separately in left and right condyle; (c) After 21 days, the
    experimental group showed an increase on sagittal dimension of the condyle. Results reflects means ± SD of 3 different ex-
    periments (p< 0.05).


    Annali di Stomatologia 2017;VIII(3):95-103                                                                                 97
    F. Perfeito et al.


    scintigraphic images were taken 7 and 21 days after               employing a low-energy high-resolution collimator.
    starting cutting, using a gamma camera (Nuclide TM                Images were acquired using a 256 × 256 × 16 matrix
    TH 22, Mediso, Hungary). This analysis does not re-               size with a 20% energy window set at 140 keV for a
    quire the animal to be killed, and can be repeated.               period of 10 min. The images were analyzed quanti-
    The animals were anesthetized with a combination of               tatively, and the radioactivity was determined in the
    ketamine (30 mg/kg) and xylazine HCl (2.5 mg/kg) in-              demarcation areas of the ROI (region of interest) in-
    tramuscularly. Aliquots (0.1 mL) containing 10,3 MBq              volving the right and left condyle on experimental and




                                                                                                            li
    of (99mTc-MDP) were injected intravenously into the               control group. The uptake of 99mTc-MDP in the re-
    tail vein of the animals. Static-planar acquisition was           gions was measured. Results were analyzed using T




                                                                                                          na
    initiated 1 h after the injection. The animals were               student test unpaired. Differences were considered
    placed in a supine position under a gamma camera                  significant when p<0.05 (Fig. 3a).




                                                                                                        io
                                                                                  az
                                                                                rn
     a
                                                             te
                                                           In
                               ni
                             io
                           iz
            Ed
 IC
C




     b
    Figure 3 a, b. Bone Scintigraphy. (a) Representative scintigraphic image for the definition of the region of interest (ROI). Red
©




    arrow is reference point to show one ROI at the condyle region. The ROI (circle) was established and the radio isotope
    count [accumulation count of 99mtechnetium-methylene-diphosphonate (99mTc-MDP)] was measured. As inferred by the
    color bar scale, red shows a high accumulation of 99mTc-MDP and blue shows a low accumulation of 99mTc-MDP; (b) Up-
    take ratio of 99mTc-MDP at the condyle region after protrusion of the mandible. The vertical axis shows the uptake ratio.
    The count rates of 99mTc-MDP in the protruded mandible condyle were significantly higher than control after 21 days. Re-
    sults express mean ± SD of 2 different experiments for each period (p< 0,05).

    98                                                                                     Annali di Stomatologia 2017;VIII(3):95-103
    Mandible protraction alters Type I collagen, osteocalcin and osteonectin gene expression in adult mice condyle


                                                                     be sure that we were dealing with osteogenic cells.
    Since our objective would be to evaluate the alter-              For immunofluorescence, we used 6 well culture flask
    Condyle cell isolation and Immunofluorescence

    ation on gene expression by osteoblasts obtained by              (Sarstedt) and platted the cells over glass cover slips
    the macerate of condyles, it was important to first              (Fisherbrand). 24 hours later, the cells were fixed
    check the cell population of this macerate. In order to          with 4% paraformaldehyde (Merk, Brazil) in phos-
    evaluate it, we first used a sequential digestion proto-         phate buffered saline (PBS) for 10 min, and washed
    col. Briefly, we dissected the condyles and freed from           three times in PBS. The cells were incubated in




                                                                                                          li
    soft tissue, cut into small pieces and rinsed in sterile         blocking solution [PBS, 1% bovine serum albumin
    phosphate-buffered saline without calcium and mag-               (Sigma), 5% normal goat serum (Sigma), 0.5% triton-




                                                                                                        na
    nesium (Sigma Aldrich). The condyle pieces were in-              X (Sigma Aldrich)] and then incubated with the follow-
    cubated with 1% trypsin-EDTA (Gibco) for 5 min, fol-             ing primary antibody: mouse antiosteopontin (R&D
    lowed by four sequential incubations with 0.2% colla-            systems) and secondary: Alexa Fluor (Invitrogen). To
    genase (Sigma Aldrich) at 37°C for 45 min each. The              evidentiate the nucleus, cells were also incubated




                                                                                                      io
    digestions produced a suspension of cells. After cen-            with propidium iodide (Sigma Aldrich) (Fig. 4).
    trifugation at 1000 g for 5 min, the pellet was resus-
    pended in 5 ml of RPMI medium (Gibco) supplement-




                                                                                az
    ed with 10% FBS (Gibco), 1% antibiotic-antimycotic               In order to evaluate differential gene expression from
                                                                     Extracting RNA from condyle in a single step

    (Gibco). The cells were seeded into 25 ml tissue cul-            cells derived from experimental and control animals,
    ture flasks (Sarstedt), and led to grow in a controlled          condyle bones were harvested from the animals, ex-
    5% CO2 95% humidified incubator at 37°C. After con-              posing them after removing muscles. Any attached
    fluence the cells were used for immunofluorescence               tissue was quickly removed using a scalpel before




                                                                              rn
    using a marker for bone forming cells (osteopontine              the condyle was immersed in liquid nitrogen. The
    OPN). OPN is produced by cells involved in bone                  samples were homogenized separately with Trizol® in
    morphogenesis such as preosteoblasts, osteoblasts,               porcelain mortar and pestle with liquid nitrogen. RNA




                                                            te
    osteoclasts, osteocytes, odontoblasts, and also hy-              was then separated and processed according to the
    pertrophic chondrocytes. So we decided to use it to              manufacturer’s protocol (Trizol ® Reagent, Life Tec-
                                                          In
                               ni
                             io
                           iz
            Ed
 IC
C
©




    Figure 4. Immunofluorescence for osteopontin. Confocal images obtained from the condyle macerate. A great number of
    cells was positive for osteopontin, indicating that they were bone forming cells. Green indicates osteopontin staining and red
    are nucleus stained by propidium iodide. Transmission image included in merged image.


    Annali di Stomatologia 2017;VIII(3):95-103                                                                                 99
    F. Perfeito et al.


    nologie, Carlsbad, California). Total RNA was quanti-       used to normalize the expression of the target gene
    fied and then treated with DNase I® (TURBO DNA-             (target gene CT mean - mean CT endogenous con-
    free Kit, Ambion® Inc., Foster, California, USA).           trol) generating ΔCT. Using the ΔCT was calculated
                                                                ΔΔCT [ΔCT sample - ΔCT calibrator (reference sam-
                                                                ple)]. Then it was applied the formula 2 -ΔΔCT for de-
    The RT for sscDNA synthesis was performed from 1 μg         termining the relative levels of expression of each tar-
    Reverse Transcriptase Reaction

    of total RNA in a final reaction volume of 20.4 μl per      get gene. The results of expression levels were




                                                                                                   li
    sample. Briefly, RNA was pre-incubated at 70° C for         launched in GraphPad Prism 5 software, for statistical




                                                                                                 na
    10 minutes with 10 pmol of each reverse primer spe-         analysis, using t student tests unpaired. Differences
    cific for the target genes, together with 10 pmol of oli-   were considered significant when p<0.05.
    go dT 18 primer, followed by storage on ice at the
    bench top. Then, was added to samples mix contain-
    ing RT buffer [250 mM Tris-HCl (pH 8.3), 375 mM
                                                                Results




                                                                                               io
    KCl, 15 mM MgCl2] and dNTP (10mM each), samples
    with this mix were incubated at 45° C for 2 min and
                                                                Sequential cut of inferior incisor generates

    then placed on ice. Finally added 1 μl of reverse tran-     The model used to generate protrusion was adapted
                                                                condyle sagittal remodeling




                                                                          az
    scriptase enzyme mix (40 U) into RT buffer [250 mM          from Tagliaro et al. (17). In our protocol we did not
    Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2] and in-         use anesthesia and the cutting using a nail plyer was
    cubated at 45°C for 1 hour along with previously de-        quick, effective and more secure than trimming using
    scribed RNA and primers. The reaction was terminat-         a dental motor, recommended by the Author. On Fig-




                                                                        rn
    ed at -20°C until used in the real time PCR. All            ure 1a we can see the generated increase on overjet.
    reagents were from Invitrogen™ (Life Technologies,          The weight gain of both groups was checked to eval-
    Carlsbad, CA, USA).                                         uate the experimental group adaptation to the in-
                                                                creased overjet. When compared to control, experi-




                                                         te
                                                                mental group showed, on the first days, a decrease
    The real time PCR was developed in 7500/ABI                 on weight gain, indicating that the animals were not
    Real time PCR

    PRISM® Sequence Detection System equipment, us-             used to the new occlusal situation leading to difficul-
                                                       In
    ing the protocol described by the reaction SYBR             ties on chewing. We can state that the functional
    Green PCR Master Mix Kit (Invitrogen™ Life Tech-            stimulation during this period decreased. After they
    nologies, Carlsbad, CA, USA). Samples in triplicate         get used to new occlusal scheme they regularized the
    were applied to 96-well plates (ABI PRISM® Optical          food intake and achieved the same weight of the con-
    96 -Well Reaction Plate with bar code Invitrogen Life       trol group at 21 days as showed on Figure 1b. The to-
                             ni

    Technologies, Carlsbad, CA, USA) in a final reaction        mographic images were taken in order to evaluate
    volume of 20 μl each. Aliquots of 1.6 μl of sscDNA          sagittal dimension alteration of the condyles from the
    sample were pipetted into each well of the plate, sub-      animals submitted to mandible protrusion, since it is
                           io


    sequently adding 18.4 μl of sybrMix [10 μl of SYBR          known that the condyle remodeling follows the direc-
    Green PCR Master Mix Kit, 1.2 μl of each primer             tion of pulling (18). It was adapted a positioner for
    (sense and antisense; 10 pmol / μl) and 6 μl sterile        anesthetized animals as showed on Figure 2a. Im-
                         iz



    filtered water]. The plate was sealed with optical ad-      ages were taken after localizing the condyle on trans-
    hesive (ABI PRISM® optical adhesive Covers, Invitro-        versal and coronal plane. The measurements were
    gen ® Life Technologies, Carlsbad, CA, USA). Real           made with 0,5 mm of accuracy (Fig. 2b). The results
            Ed




    time PCR reactions occurred in the following thermal        showed that mandible protrusion was able to gener-
    cycle: [stage 1] a cycle of 50° C/2 min; [stage 2] cycle    ate sagittal condyle posterior remodeling on adult ani-
    at 95°C/10 min; [stage 3] 40 cycles of 95°C/0.15 min,       mals, when compared to no protruded controls
    followed the dissociation curve from 60˚C to analyze        (p<0.05) (Fig. 2c).
    the specificity of the amplicons. The mRNA value for
    each gene was normalized relative to the housekeep-
 IC




    ing mouse S26 mRNA levels in RNA samples. Primer
                                                                Mandible protrusion leads to increase on condyle

    sequences (forward and reverse) were as follows:
                                                                cellular activity and enhanced expression of Type

     S26, 5`- CGTGCTTCCCAAGCTCTATGT -3` and 5` -                On the scintigraphy, the selected regions of interest
                                                                I collagen, osteocalcin and osteonectin

    CGATTCCTGACAACCTTGCTATG -3`;                                (ROI) on condyle were evaluated in both sides of ex-
C




    Bglap3, 5`-CTTGGTGCACACCTAGCAGA -3` and 5`-                 perimental and control animals (Fig. 3a). The plotted
    ACCTTATTGCCCTCCTGCT -3`;                                    results indicated that on 7 days it was not possible to
    Col1a1, 5`- GCTCCTCTTAGGGGCCACT -3` and 5`-                 register a statistical difference of 99mTc-MDP capita-
    ATTGGGGACCCTTAGGCCAT -3`;                                   tion comparing experimental and control groups. But
©




    Sparc, 5`- AAACATGGCAAGGTGTGTGA -3` and 5`-                 after 21 days it was possible to see a statistical rele-
    AAGTGGCAGGAAGAGTCGAA-3`.                                    vant increase on the capitation by the condyles sub-
    For relative quantification was performed a compara-        mitted to traction by protrusion (p<0.05) (Fig. 3b),
    tive analysis of the expression of target transcripts       corroborating the results obtained by the tomography.
    genes versus endogenous control using the compara-          After isolation and expansion of the cell population
    tive CT method, which the endogenous control was            obtained from sequential digestion of condyle macer-

    100                                                                           Annali di Stomatologia 2017;VIII(3):95-103
    Mandible protraction alters Type I collagen, osteocalcin and osteonectin gene expression in adult mice condyle


    ate, it was possible to show that a great number of            Since condyle remodeling occurs due to endochon-
    bone forming cells were present on the population.             dral ossification, the majority of the works have
    Osteopontin antibody stained the majority of the cells         been trying to investigate the chondrogenesis (8,
    as shown in Figure 4, indicating that the condyle              25, 26). We focused, instead, on the next step of
    macerate could be used for total RNA extraction to             bone formation, trying to investigate if the functional
    evaluate differential gene expression of bone forming          stimulation of mandible would lead to different ex-
    markers. The expression of Type I collagen, osteocal-          pression of bone forming markers by condyle os-




                                                                                                       li
    cin and osteonectin by the bone forming cells isolated         teoblasts. We choose 3 specific bone markers to be
    from the condyle were affected by the mechanical




                                                                                                     na
                                                                   evaluated. Since collagen Type I gene expression
    stimulation. During the first days, when the animals           occurs during all process of bone formation (27), it
    from experimental group were not able to chew prop-            was our first choice. osteocalcin and osteonectin are
    erly we observed a decrease on the expression of the           also well known specific markers for osteogenesis
    three genes compared to control animals. After they            (28, 29). Using primers for these 3 genes, we per-




                                                                                                   io
    start to chew normally and had to protrude in order to         formed a Real-time PCR and found that all of them
    compensate the increased overjet, there was a recov-           had the expression level diminished when the ex-
    ering on the expression of these genes allowing at 21          perimental animals were not able to chew, indicating




                                                                              az
    days no statistical difference on their level (p<0.05)         that even in adults, the maintenance of adequate
    (Fig. 5a-d). As soon as the animals started to in-             functional stimulation is necessary to keep the regu-
    crease the condyle traction, due to mandible protru-           lar level of gene expression. On the other hand, the
    sion, gene expression pattern of Type I collagen, os-          functional stimulation enhancing, generated by




                                                                            rn
    teocalcin and osteonectin increased.                           condyle traction, after the animals get used to big
                                                                   overjet, lead to an increase on osteogenic markers
                                                                   gene expression level.
                                                                   Type I collagen synthesis is of particular interest.




                                                          te
    Discussion
                                                                   The synthesis of this collagen is closely regulated,
    Little is known about the mechanisms involved on               since deficiencies or excess of the protein can
    mandible remodeling under functional stimulation. In           cause serious disorders (30). Osteonectin, recently,
                                                        In
    a recent integrative review conducted by our group             appears as a promising target for preventing or
    (19) we found only 15 relevant works dealing with this         treating bone diseases (31). Osteocalcin, since last
    subject in the last 20 years. It is well known that func-      decade, is recognized as an important regulator of
    tional stimulation interferes with condyle remodeling          bone mechanotransduction (32-34). So, knowing
    during growing periods (17, 20-22). However, it re-            that functional stimulus interferes so much on Type
                               ni

    mains unclear the role of functional stimulus on adult         1 collagen, osteonectin and osteocalcin expression
    condyle remodeling. Our adapted protrusion model               opens new frame for therapeutic approaches, not
    allowed the investigation of condyle remodeling in             only related to mandible retrusion but also to tem-
                             io


    adult mice. On the beginning the animals diminished            poromandibular joint disorders.
    the food intake. The difficulty on chewing demonstrat-         Together, our results corroborated previous findings
    ed by the animals immediately after the initial cutting        and expanded them, now showing that the synthesis
                           iz



    is in accord to orofacial proprioception neurophysiolo-        of Type I collagen, osteocalcin and osteonectin can
    gy. It is known that body needs an adaptation period           have the expression pattern altered by changes on
    when changes in occlusal scheme occurs. It gener-              functional stimulation, in adults. These current ob-
            Ed




    ates a delay on regular food intake (23). So, our ex-          servations highlight the needing for further investi-
    perimental group delayed to get used to increased              gations.
    overjet and due to that diminished the food intake.
    But the curve of weight gain (Fig. 1b) indicated that
    the experimental group after recovering the capacity
    of regular chewing, even having to protrude, was able
                                                                   Conclusion
 IC




    to equalize the weight gain with the control group. It
                                                                   Functional stimulation is necessary to maintain the
    indicates that the animals learned how to forward the
                                                                   regular gene expression of collagen, osteocalcin
    mandible in order to chew, making our model ade-
                                                                   and osteonectin by condyle bone forming cells, in
    quate for the study. The tomographic study demon-
                                                                   adult mice. Protraction of mandible generates alter-
C




    strated that adult mice showed change on condyle
                                                                   ation on these genes expression by condyle cells.
    shape when submitted to mandible forwarding. It was
    also observed by scintigraphic study a higher cellular
    activity when compared to control (Fig. 2, 3). It is in
    accord with a previous study, conducted in rats, where
©




                                                                   Aknowledgements
    the changing of occlusal scheme, by removing some
                                                                   We would like to thank the ARCATA clinic staff for
    teeth, lead to alteration on condyle shape (24). Our re-
                                                                   the kind help with the tomography. This work was
    sults reinforced the effect of functional stimulus on
                                                                   supported by CAPES, CNPq and FAPEMIG grants.
    condyle shape and cell activity even being an adult.



    Annali di Stomatologia 2017;VIII(3):95-103                                                                        101
    !

    F. Perfeito et al.
    !

    !
                                                                                                                                                                                                     $

                                                                                                                                              !"#$                                                                                                                                                                                                                                                                                                                                                                             ! " #$ %
        + , - ( * 9 9 .% & 6% :6/ ; < = 6! % )$ 0 $                                                                   4 21                                                                                                                                                                                                               4 10




                                                                                                                                                                                                                             , - % * + 8 8 .' ( 6' 96/ : ; < 6! " #$ %
                                                      6& % ( / 0 ).> * 7 6? 8 6@ 4 A




                                                                                                                                                                                                                                                                         6( ' * / $ #.= + 7 6> ? 6@ 4 A
                                                                                       B 0 ( ? .' ( 0 ( 8 6C & .' D
                                                                                                                      $ 23




                                                                                                                                                                                                                                                                                                          B $ * > .) * $ * ? 6C ( .) D
                                                                                                                                                                                                                                                                                                                                         3 12




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    li
                                                                                                                                                                & " ' (' )
                                                                                                                      $ 21
                                                                                                                                                                       %                                                                                                                                                                 3 10
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   " # $ %$ &




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  na
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  !
                                                                                                                      1 23                                                                                                                                                                                                               0 12
                                                                                                                                !"#                                 !"$                                                                                                                                                                                                                                                  '#(                                                                                                                                                                                                                                                  '#(
                                                                                                                      1 21                                                                                                                                                                                                               0 10
                                                                                                                             ! % & ' ( % )*                + , - * ( ./ * & ' 0 )




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                io
                                                                                                                                                                                                                                                                                                                                                                                                           & ' ( ) * ' #+                                                                                                                                                                                    , - % + * ./ + ( ) $ #
                                                                                                                                              5 67 0 8 9                                                                                                                                                                                                                                                                                                                                                                                 5 68 $ 9 :
                                                                                                                                                                                      !                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      $
                                                                                                                                               ! " #$                                                $
    !                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          ! " #$ %
                                                                                                                                                                                                     $




                                                                                                                                                                                                                                                                                                                                     az
        7 * + ( ) 6 6 ," & 3" 83- 9 : ; 3! " #$ . $




                                                                                                                      2 01
                                                                                                                                                                                                                                                                                                                                         0 15




                                                                                                                                                                                                                             + , % * - : : .' ( 7' ;7/ < = > 7! " #$ %
    !
                                                      3& " ( - . #,< ) 4 3= 5 3> 2 ?




                                                                                                                      2 0/
                                                                                       @ . ( = ,' ( . ( 5 3A & ,' B




                                                                                                                                                                                                                                                                         7( ' * / $ #.? - 8 7@ 9 7A 2 4
                                                                                                                                                                                                                                                                                                          B $ * @ .) * $ * 9 7C ( .) D
                                                                                                                                                                                                                                                                                                                                         0 14
                                                                                                                      $ 01




                                                                                                                                                                                                                                                                                                                                   rn
                                                                                                                      $ 0/                                                                                                                                                                                                               0 13


                                                                                                                      / 01
                                                                                                                                                                                                                                                                                                                                         0 12




                                                                                                                                                                                        te
                                                                                                                      / 0/
                                                                                                                              % " & '(" #                    ) * + ) ( ,- ) & ' . #                                                                                                                                                      0 10
                                                                                                                                                                                                                                                                                                                                                                                                                    & ' ( )*' #                                                                                                                                                                              + , % - * ./ - ( ) $ #
                           a                                                                                                                  2 $ 34 . 5 6                                                          b
    $
                                                                                                                                                                                      In                                                                                                                                                                                                                                                                                                                                2 6 78 $ 9 :


                                                                                                                                              ! " # $%
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            ! " # $# %
                                                                                                                      4 10
        , - " $ + 9 9 .' ( 6' : 6/ ; < = 6! " # $ %




                                                                                                                                                                                                                                                                                                          >%#
                                                                                                                                     ni                                                       & ' ( ) * + + ,- . /0* 1 * 0
                                                      6( ' $ / # *.> + 7 6? 8 6! 4 @
                                                                                       A # $ ? .) $ # $ 8 6B ( .) C




                                                                                                                      3 12
                                                                                                                                                                                                                                                    2 3 /- 4/5 - . 6) - 07




                                                                                                                                                                                                                                                                                                                                                                                                                                                      !!                                                                                                                                           !!!                                                                                                                                                                                                      !!
                                                                                                                                                                                      !
                                                                                                                                   io

                                                                                                                      3 10                                      & " ' (' )                                                                                                                                >##                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                $
    !                                                                                                                                                                  %
                                                                                                                      0 12
                                                                                                                                 iz



                                                                                                                                !"#                                 !"$                                                                                                                                                          %#
                                                                                                                      0 10
                                                                                                                             & ' ( ) $ ' *+                , - " + $ ./ + ( ) # *
                                                                                          Ed




                                                                                                                                              5 67 # 8 9
                                                                                                                                                                                      $
                                                                                                                                                                                                                                                                                                                                         #
                                                                                                                                                                                                                                                                                                                                                                                 8 - . 6 ) - 0/= > /: ; <+ /8 - 0
                                                                                                                                                                                                                                                                                                                                                8 - . 6 ) - 0/9 /: ; <+ /8 - 0




                                                                                                                                                                                                                                                                                                                                                                                                                                                                & ' ( * ) ,? * . 6 ; 0/= > /: ; <+ /8 - 0




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       8 - . 6 ) - 0/= > /: ; <+ /B ( ; ) 5
                                                                                                                                                                                                                                                                                                                                                                                                                      & ' ( * ) ,? * . 6 ; 0/9 /: ; <+ /8 - 0




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                8 - . 6 ) - 0/= > /: ; <+ /@ A 0; (




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  8 - . 6 ) - 0/9 /: ; <+ /B ( ; ) 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            8 - . 6 ) - 0/9 /: ; <+ /@ A 0; (




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             & ' ( * ) ,? * . 6 ; 0/= > /: ; <+ /B ( ; ) 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   & ' ( * ) ,? * . 6 ; 0/= > /: ; <+ /@ A 0; (




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              & ' ( * ) ,? * . 6 ; 0/9 /: ; <+ /B ( ; ) 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      & ' ( * ) ,? * . 6 ; 0/9 /: ; <+ /@ A 0; (




                                                                                                                                              ! " # $%
    $
                                                                                                                      3 12
        + , " $ - 8 8 .' ( 5' 9 5/ : ; < 5! " # $ %
                                                      5( ' $ / # *.= - 6 5> 7 5! 4 ?
                                                                                       @ # $ > .) $ # $ 7 5A ( .) B




                                                                                                                      3 10
 IC




                                                                                                                      0 12
C




                                                                                                                      0 10                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       !
                                                                                                                             & ' ( )$' *                   + , " - $ ./ - ( ) # *
                           c                                                                                                                                                              !                         d
                                                                                                                                              4 3 56 # 7 8
©




    Figure 5. Real-time PCR. Diferential expression of: (a) Collagen (Col1), (b) Osteocalcin (Bglap) and (c) Osteonectin (Sparc)
                                                                                                                                                                                          !

    evaluated at 7 days and 21 days. All of them showed lower expression on experimental group at 7 days, compared to con-
    trol (p<0.05). On 21 days no statistical difference on gene expression was observed (p<0.05), indicating that the experimen-
    tal group showed an enhance on gene expression during the period from 7 to 21 days; (d) Differential expression of Type I
    Collagen, Osteocalcin and Osteonectin by experimental group  $ analyzed in terms of control % (p<0.05).




    102                                                                                                                                                                                                                                                                                                                                                                                                             Annali di Stomatologia 2017;VIII(3):95-103
    Mandible protraction alters Type I collagen, osteocalcin and osteonectin gene expression in adult mice condyle


                                                                                   (2):79-83.
                                                                               19. Valerio P, Macedo FJM, Simoes WA. Remodelative alter-
    References

    1.    Joshi N, Handam A, Fakhouri WD. Skeletal malocclusion:                   ations of TMJ with the use of protraction appliances: an in-
          a developmental disorder with a life long morbidity. J Clin Med          tegrative review. Ortodontia SPO. 2014;47(3):218-223.
          Res. 2014;6(6):399-308.                                              20. Watahiki J, Yamaguchi T, Irie T, Nakano H, Maki K,
    2.    Baume RM, Buschang PH, Weistein S. Stature, head                         Tachikawa T. Gene expression profiling of mouse condylar
          height and growth of the vertical face. Am J Orthod. 1983;               cartilage during mastication by means of laser microdissection




                                                                                                                        li
          83:477-483.                                                              and cDNA array. J Dent Res. Mar 2004;83(3):245-249.
    3.    Arvystas MG. The rationale for early orthodontic treatment.          21. Shibukawa Y, Young B, Wu C, Yamada S, Long F, Pacifi-




                                                                                                                      na
          Am J Orthod Dentofacial Orthop. 1998;113:15-18.                          ci M, Koyama E. Temporomandibular joint formation and
    4.    Rabie AB, Al-Kalaly A. Does the degree of advancing dur-                 condyle growth require Indian hedgehogsignaling. Dev
          ing functional appliance therapy matter? Eu J Orthod.Jun                 Dyn. Feb 2007;236(2):426-434.
          2008;30(3):274-282.                                                  22. Liu YD, Liao LF, Zhang HY, Lu L, Jiao K, Zhang M, Zhang
    5.    Shibata S, Fukuoka H, Sato R, Abe T, Suzuki Y. 2012. An                  J, He JJ, Wu YP, Chen D, Wang MQ. Reducing dietary load-




                                                                                                                    io
          in situ hybridization study of the insulin-like growth factor sys-       ing decreases mouse temporomandibular joint degradation
          tem in developing condylar cartilage of the fetal mouse                  induced by anterior crossbite prosthesis. Osteoarthritis
          mandible. Eur J Histochem. May 2008;2356(2):e23.                         Cartilage. Feb 2014;22(2):302-312.
    6.    Liu Q, Gibson MP, Sun H, Qin C. Dentin sialophosphopro-              23. Kumar A, Grigoriadis J, Trulsson M, Svensson P, Svensson




                                                                                           az
          tein (DSPP) plays an essential role in the postnatal devel-              KG. Effects ofshort-term training on behavioral learning and
          opment and maintenance of mouse mandibular condylar car-                 skill acquisition during intraoralfine motor task. Neuro-
          tilage. J Histochem Cytochem. Oct 2013;61(10):749-758.                   science. Jul 2015;7;306:10-17.
    7.    Morimoto H, Baba R, Haneji T, Doi Y. Double-stranded RNA-            24. Yokoyama M, Atsumi T, Tsuchiya M, Koyama S, Sasaki K.
                                                                                   Dynamic changes in bone metabolism in the rat temporo-




                                                                                         rn
          dependent protein kinase regulates insulin-stimulated chon-
          drogenesis in mouse clonal chondrogenic cells, ATDC-5. Cell              mandibular joint after molar extraction using bone scintigraphy.
          Tissue Res. Jan 2013;351(1):41-47.                                       Eur J Oral Sci. Aug 2009;117(4):374-379.
    8.    Enomoto A, Watahiki J, Nampo T, Irie T, Ichikawa Y, Tachika-         25. Cohen WA, Servais JM, Polur I, Li Y, Xu L. Articular carti-
                                                                                   lage degeneration inthe contralateral non-surgical tem-




                                                                      te
          wa T, Maki K. 2014. Mastication markedly affects mandibu-
          lar condylar cartilage growth, geneexpression, and mor-                  poromandibular joint in mice with a unilateral partial dis-
          phology. Am J Orthod Dentofacial Orthop. Sep 2013;146                    cectomy. J Oral Pathol Med. Feb 2014;43(2):162-165.
          (3):355-363.                                                         26. Jahan E, Matsumoto A, Rafiq AM, Hashimoto R, Inoue T,
                                                                    In
    9.    Serrano MJ, So S, Hinton RJ. Roles of notch signalling in                Udagawa J, Sekine J, Otani H. Fetal jaw movement affects
          mandibular condylar cartilage. Arch Oral Biol. Jul 2014;59(7):           Ihh signaling in mandibular condylarcartilage development:
          735-740.                                                                 the possible role of Ihh as mechanotransduction mediator.
    10.   Umeda M, Terao F, Miyazaki K, Yoshizaki K, Takahashi I.                  Arch Oral Biol. Oct 2014;59(10):1108-1118.
          2015. MicroRNA-200ª Regulates the Development of                     27. Harris SE, Bonewald LF, Harris MA, Sabatini M, Dallas S,
                                   ni

          Mandibular Condylar Cartilage. J Dent Res. Jun 2014;94(6):               Feng JQ, Ghosh-Choudhury N, Wozney J, Mundy GR. Ef-
          795-802.                                                                 fects of transforming growth factor betaon bone nodule for-
    11.   Bollen AM, Makinen KK, Makinen PL, Carlson DS. Col-                      mation and expression of bone morphogenetic protein 2, os-
          lagenolytic and phosphatase activity in the rat mandible af-             teocalcin, osteopontin, alkaline phosphatase, and type I col-
                                 io


          ter functional protrusion. Arch Oral Biol. 1989;34(4):267-273.           lagen mRNA inlong-term cultures of fetal rat calvarial os-
    12.   Chen D, Wang X, Luo S. Histologic and histochemical stud-                teoblasts. J Bone Miner Res. Jun 1994;9(6):855-863.
          ies on the temporomandibular articular disc in young grow-           28. Brown JP, Delmas PD, Malaval L, Edouard C, Chapuy MC,
                               iz



          ing rats after functional mandibular advancements. Hua Xi                Meunier PJ. Serum boneGla-protein: a specific marker for
          Kou Qiang Yi XueZaZhi. 1997;15(4):343-345.                               bone formation in postmenopausal osteoporosis. Lancet. May
    13.   Lou X, Chen Y, Luo S, Chen G. Circadian rhythm changes                   1984;191(8386):1091-1093.
                                                                               29. Shimokawa H. Osteonectin, a new protein. Nihon Shika Ishikai
             Ed




          of the levels of endogenous insulin in growing rat condylar
          cartilage after functional mandibular protrusion. Hua Xi Kou             Zasshi. 1984;37(5):487-491.
          Qiang Yi XueZaZhi. 2000;18(4):252-254.                               30. Marini J, Smith SM. Osteogenesis Imperfecta. 2015; Apr 22.
    14.   Shen G, Rabie AB, Hägg U, Zhao Z. Expression of type X                   In: De Groot LJ,Beck-Peccoz P, Chrousos G, Dungan K,
          collagen in condylar cartilage during mandibular protrusion.             Grossman A, Hershman JM, Koch C, McLachlanR, New M,
          Hua Xi KouQiang Yi XueZaZhi. 2000;18(2):78-84.                           Rebar R, Singer F, Vinik A, Weickert MO, editors. Endotext
    15.   Xiong G, Chen Y, Ye L, Lou S. Circadian rhythm changes                   [Internet]. South Dartmouth (MA): MDText.com, Inc.
                                                                               31. Ribeiro N, Sousa SR, Brekken RA, Monteiro FJ. Role of
 IC




          of the levels ofendogenous insulin in growing rat’s blood af-
          ter functional mandibular protrution. Hua Xi Kou Qiang Yi                SPARC in bone remodeling and cancer-related bone metas-
          XueZaZhi. 2000;18(5):349-351.                                            tasis. J Cell Biochem. Jan 2014;115(1):17-26.
    16.   Ye L, Chen Y, Luo S. Expression of mRNA of osteocalcin in            32. Kreke MR, Goldstein AS. Hydrodynamic shear stimulates os-
          ondylar cartilage of young SD rats after functional protrusion.          teocalcin expression but not proliferation of bone marrow stro-
C




          Zhonghua Kou Qiang Yi XueZaZhi. 2001;36(6):404-407.                      mal cells. Tissue Eng. May-Jun 2004;10(5-6):780-788.
    17.   Chen J, Sobue T, Utreja A, Kalajzic Z, Xu M, Kilts K, Young          33. Iqbal J, Zaidi M. Molecular regulation of mechanotransduc-
          M. Sex Differences in Chondrocyte Maturation in the                      tion. Biochem BiophysRes Commun. Mar 2005;18;328(3):
          Mandibular Condyle from a Decreased Occlusal Loading Mod-                751-755.
                                                                               34. Wang B, Liu YY, Zheng JB, Yuan SX, Chen GX. Changes
©




          el. Calcif Tissue Int. August 2011;89(2):123-129.
    18.   Simoes WA, Petrovic A, Stutzmann J. Modus operandi                       of osteocalcin and IGF-I during bone lengthening. Chin J Trau-
          of Planas’ appliance. J Clin Pediatr Dent Winter. 1992;16                matol. Jun 2005;8(3):151-515896271.




    Annali di Stomatologia 2017;VIII(3):95-103                                                                                                103
