## 4 Discussion  
Unilateral posterior crossbite is a malocclusion disease characterized by an inverse relationship between the lower and upper dental cusps [1; 13; 14; 15; 16]. Electrophysiological studies have demonstrated that, when chewing on the affected side, the ipsilateral muscle shows reduced activity and the contralateral muscle activity is normal or increased [2; 17; 18; 19]. The increased work of the contralateral muscle depends on trying to compensate for the affected side. All changes in muscle function determine the morphological changes that seem to correlate with the hypertrophic and atrophic responses of contralateral and ipsilateral muscles, respectively [6; 7; 8; 20; 21]. Although several functional and morphological data support the existence of hypertrophy and hypertrophy or atrophy [22], no investigations about masseter muscle fiber diameters during unilateral posterior crossbite exist in the literature. The aim of the present study was to perform a measurement of fiber diameter, both for both the ipsilateral and contralateral sides, using immunofluorescence pictures and ImageJ software. Immunofluorescence results showed the expression of gamma sarcoglycan along the sarclemma and within fibers in the control muscle, and both in contralateral and ipsilateral masseter muscles. The intensity of fluorescence was higher in the control muscles and in the contralateral muscles than in the ipsilateral muscles, supporting our previous results that showed a higher expression of sarcoglycans and integrins in contralateral masseter muscles when compared to ipsilateral ones [6]. Sarcoglycans and integrins are proteins systems that play a crucial role in sarcolema stabilization during muscle contraction. As such, their increase or decrease correlate with a good or impaired muscle activity, respectively [23; 24; 25; 26]. Despite this, the fluorescence staining pattern was good for marking the fibers of all muscles in order to perform diameter measurements. It is widely accepted in the literature that a hypertrophic adult muscle has a larger fiber diameter than a normal muscle. Hypertrophy in contralateral masseter muscle has been indirectly linked to the existence of a higher number of myonuclei and satellite cells. The higher workload would cause the satellite cells to proliferate and donate myonuclei to pre-existing fibers, determining the hypertrophy of muscle; moreover, the high expressions of sarcoglycans and integrins are explained by the myonuclear domain theory: more myonuclei results in more muscle protein synthesis. The expression of myogenesis markers in contralateral muscle has also been determined, supporting that new fiber formation takes place. In contrast, a hypertrophic muscle has very thin muscle fibers due to the loss of contractile elements; this loss become more severe in atrophy. The hypotrophic condition  
Figure 6: Compound panel of Gaussian curves showing the normal distribution of fiber diameters and cross-sectional area values for the control and for both ipsilateral and contralateral muscles of the crossbite group. in the ipsilateral masseter muscle were indirectly demonstrated by a low number of myonuclei and satellite cells; moreover, both sarcoglycans and integrins were found to be reduced in ipsilateral muscle and could also be explained by the myonuclear domain theory. In atrophic masseter muscle, it was also found to be increased in the extracellular matrix. All these morphological data represent an indirect way to establish the existence of hypertrophic and/or hypotrophic/atrophic responses in masseter muscle. Through radiological investigation, it is possible to establish the entire muscle volume for both sides. Despite this, the aforementioned methods do not allow to know whether the increase in volume depends on an increase of fiber thickness or on new fiber formation; they also do not allow to know the diameter of individual fibers.