## 3 Results and Discussion  
Immunofluorescence results showed that during unilateral posterior crossbite the contralateral muscle, if compared to the ipsilateral one, has: (1) an increased expression of Laminin; (2) increased expression of Collagen IV; (3) increased expression of MMP-9; and (4) the same expression of MMP-2. Laminin is an extracellular matrix protein that interacts with integrins and dystrophin glycoprotein complex representing an important interactor between muscle fibres cytoskeleton and extracellular matrix [22,23]. Our data show the typical Laminin staining pattern around the muscle fibres, at basal lamina level (Figure 1). Moreover, results show that this protein is more expressed in the contralateral muscle fibres then ipsilateral ones (Figure 1A,B), and this difference is statistically significant as shown in Figure 5. That is in accordance with our previous results showing an increased sarcoglycans and integrins expression in the contralateral side if compared to the ipsilateral side [7,8]. From this, it is possible to speculate that in the contralateral side the increased of muscle proteins determines in turns the increase of their extracellular matrix interactor. Muscle hypertrophy is often accompanied by increased collagen deposition [24,25]. The basement membrane collagens directly surround muscle fibres and assist in the transfer of lateral and longitudinal transmission of force in muscle [24]. Our results show an increase in collagen IV expression in the contralateral muscle if compared to the ipsilateral one (Figure 2), and this difference is statistically significant as shown in Figure 5. That is in accordance with the evidence of hypertrophic response of contralateral muscle. In the ipsilateral side it is possible to observe collagen IV staining pattern. This result suggest that the ipsilateral muscle is not under a manifest hypotrophy/atrophy condition. Changes in the basement membrane collagens were accompanied by generally similar changes in the gelatinases MMP2 and MMP9 [24; 25]. Metalloproteinases are proteins with enzymatic activity that play a key role in selective proteolysis of ECM components [26; 27]. MMP2 and MMP9 are the typical MMPs of skeletal muscle and are involved in the degradation of collagen IV [28]. In the contralateral muscle it is possible to observe a MMP2 and MMP9 staining pattern. These metalloproteinases are both around and within muscle fibres. In the ipsilateral side the MMP2 expression is similar to the contralateral side (Figure 3) and this difference is not statistically significant as shown in Figure 5; the MMP9 staining pattern is weak if compared to the contralateral side (Figure 4), and this difference is statistically significant as shown in Figure 5.  
Figure 1: Immunofluorescence reactions anti Laminin (red fluorescence) in contralateral (**A**) and ipsilateral (**B**) masseter muscle of patients affected by unilateral posterior crossbite. The fluorescence pattern of laminin is mainly located around the muscle fibres, at basal lamina level. The contralateral muscle seems to show a higher Laminin staining pattern if compared to the ipsilateral. \(p\)-value \(<\) 0.05. Figure 2: Immunofluorescence reactions anti collagen IV (red fluorescence) in contralateral (**A**) and ipsilateral (**B**) masseter muscle of patients affected by unilateral posterior crossbite. The fluorescence pattern of collagen IV seems to be a higher fluorescence pattern if compared to the ipsilateral one. \(p\)-value \(<\) 0.05. MMP2 is always expressed in skeletal muscle and does not undergo particular regulation processes; MMP9, instead, has been found increased in response to high workload [29]. Studies have also demonstrated that high levels of MMP9 leads to hypertrophy and to an increased muscle strength. Our results, showing a higher expression of MMP9 in the contralateral muscle are in accordance with evidence of hypertrophic response of contralateral masseter muscle.