Introduction
Patellofemoral pain syndrome (PFPS) is often unreasoned knee anterior pain. Its prevalence is 25% in the general population and is more common in young and active people [1]. Although patellar maltracking in PFPS patients can be due to a variety of reasons, one of the common signs associated with this disorder is the lower activity of vastus medialis oblique (VMO) than vastus lateralis muscle (VL) [2]. The VMO is located at an angle of 45° to 50° to the longitudinal axis of the femur on the medial side of the patella. The most important function of this muscle is as a dynamic stabilizer of the patella to prevent excessive patellar lateralization [3]. Dynamic stability of the patella is mainly performed by the VMO and VL muscles, especially VMO [4]. Therefore, strengthening VMO can effectively improve the function of people with PFPS.
Regarding therapeutic management, most researchers agree that non-surgical and non-pharmacological interventions are first-line treatments for PFPS. An effective and non-invasive method for these individuals is physiotherapy, including general strengthening of the quadriceps, muscular stretching, patellar taping, and specific VMO strengthening [5]. The muscular imbalance between VMO and VL may lead to patellar lateralization, causing PFPS. Therefore, attention to specific VMO strengthening is crucial [6]. In previous studies, different exercises have been introduced to specifically strengthen VMO, including open and closed kinetic chain exercises [7], changes in the tibia [8] and hip rotations [9], adding ankle dorsiflexion during SLR exercise [10] and hip adduction [11]. One of the exercises suggested for VMO activation is the SLR maneuver with different angles of hip rotation. Sykes et al. (2003) showed that SLR with external hip rotation could increase VMO electrical activity relative to VL [9]. Although other studies have demonstrated that SLR with external hip rotation cannot be used as a specific exercise to activate VMO, there is still controversy [12]. On the other hand, another study mentioned that adding contraction of ankle dorsiflexor muscles during SLR exercise can effectively activate VMO and VL [10, 13].
While performing the SLR maneuver in the supine position, the activity of the rectus femoris muscle is dominant over the VMO muscle due to hip flexion [14]. To our knowledge, no study has been performed on VMO muscles to create more activity while performing the SLR maneuver sitting. Therefore, because the rectus femoris suffers from active insufficiency in the sitting position [15], it can be expected that by creating this change, the activity of other parts of the quadriceps muscle will be more dominant than the rectus femoris. 
In recent years, ultrasonography has been widely used to measure morphological changes in skeletal muscle, such as thickness and fiber angle [16]. Compared to existing imaging methods, ultrasonography is a method with high validity and reliability to examine the relationship between the strength and size of muscles [17]. Therefore, in this study, ultrasonography was used to record changes in the thickness and fiber angle of the knee muscles.
Regarding the importance of VMO strengthening in patients with PFPS and disagreement about exercises that specifically activate VMO, this study measured the thickness and fiber angle of VMO and VL muscles during SLR exercise in different positions of this maneuver in individuals with and without PFPS. These positions included performing SLR exercises with maximum muscle contraction in a sitting position by combining different hip rotations with and without maximal contraction of the ankle dorsiflexor muscles. 
Materials and Methods 

Study participants
This quasi-experimental study was performed on 40 volunteers in two groups. One group included 20 healthy individuals (13 males and 7 females) with a mean age of 23±2.1 years, a height of 170±6 cm, and a weight of 64.65±3.61 kg. The inclusion criteria for healthy individuals were as follows: no history of knee pain in the last three months before the study, no record of specific pathology in the lower limbs, and no pain in performing more than two activities of running, jumping, sitting for a long time, and going up and down the stairs. Another group consisted of 20 individuals with PFPS (13 males and 7 females) with a mean age of 22.75±3.43 years, a height of 169±8 cm, a weight of 64.1±4.63 kg, and no history of knee and hip trauma. The inclusion criteria in the PFPS group included pain in the front of the knee and around the patella during at least two activities previously mentioned, knee pain for at least the past three months felt for most days, pain in the medial and lateral facet of the patella [18, 19]. The exclusion criteria were a history of lower limbs surgery in the past 12 months before the study, previous musculoskeletal injuries of the hip, knee, or ankle, inflammatory and swelling conditions in the knee, and a history of dislocation or subluxation of the patella [18]. 
PFPS individuals were selected by simple non-random sampling from patients referred to the physiotherapy clinic of Shahid Beheshti University of Medical Sciences and healthy individuals from healthy students of that university. To determine the sample size, a pilot study was performed on 10 cases in each group with α=0.05 and a power of 80%. Finally, 20 subjects in each group were calculated. In this project, all participants signed written informed consent. Individuals also completed a demographic information questionnaire. 
 Visual analog scale (VAS) was used to assess pain in people with PFPS, and they were asked to show their pain intensity on the pain ruler from 0 to 10. Due to the evaluation of maximal isometric contraction in the present study, individuals with moderate pain intensity (VAS between 3 and 6) were included.

Study procedure 
To evaluate the interrater reliability of sonographic measurements, including VMO and VL muscle thickness and fiber angle, a preliminary study was performed on 10 healthy individuals to replicate these parameters during maximal SLR contraction in the different positions. An examiner conducted the measurements for 1 week, and the mean values ​​with the three-time measurements were used to calculate the intraclass correlation coefficient (ICC). 
After confirming the interrater reliability, the researcher performed all VMO and VL muscle thickness measurements and fiber angle measurements at the resting position through ultrasonography. Then, before conducting the main test, the subjects received a warm-up period by walking at their normal speed for 5 min on a treadmill and stretching the quadriceps, hamstrings, calf, and hip adductor muscles. Muscle stretching was done for 30 s, and three repetitions of the stretch for each muscle [20]. These procedures were performed on participants by the same physiotherapist. After preparing the participants, the person was placed in a chair designed for this study so that the subject was sitting with straight knees and ankles in a neutral position (Figure 1). Individuals were then asked to randomly perform different positions of SLR maneuver with their maximum strength in 6 positions: internal, external, and neutral hip rotations with or without contraction of ankle dorsiflexor muscles. To perform different hip rotations, the people rotated their hips so that the axis designed on the device, on which the people’s feet rested, was parallel to the thumb toe. The axes were designed at a 45° angle on both sides, and participants were asked to create maximum internal and external rotations of their hips. Next, the people applied their full strength to the load cell in front of the ankle and held the contraction for at least 5 s. Performing SLR in a sitting position can lead to more activity of VMO and VL rather than the rectus femoris. 
All VMO and VL muscle thickness measurements and fiber angles were performed in this position after ensuring the lower limb was stable during the SLR maneuver. Ultrasound imaging was performed by the Sonography Capture software so that the software regularly recorded ultrasound images during the 5 seconds when the participants completed their SLR maneuver. This software could perform ultrasonographic measurements of the target muscles when the person had created the most torque. The participant repeated each SLR maneuver 3 times at 1-minute intervals. When the measures related to the first 6 positions were finished, the person rested for 4 min. The same procedure was followed for the other 5 SLR maneuvers randomly selected. Dependent variables included VMO and VL thickness at rest and contraction positions, VMO and VL fiber angle at rest and contraction positions, and the ratio of VMO to VL thickness at different contraction positions.
This study used the ultrasonography device (Honda Co, Japan) with a frequency of 7.5 MHz and a 5-cm linear probe to measure the VMO and VL muscle thickness and fiber angle. To calculate the thickness of the VMO muscle, we placed the probe of the ultrasound device, after its soaking in some gel, horizontally in the supra medial of the patella when we ensured that the lower limb was fixed. Then, we moved the probe along the medial side of the patella toward the proximal and distal to reveal the VMO fibers. After ensuring that the VMO muscle image appeared on the monitor, we saved the image. The maximum distance between the anterior and posterior fascia of the muscle was considered as its thickness [21]. To measure the VMO fiber angle, we placed the ultrasound probe parallel to the muscle fibers so that VMO fibers appeared parallel to each other in the ultrasound image. In this case, the angle formed between the longitudinal axis of the ultrasound probe and the line connecting the anterior superior iliac spine to the patella center was considered a VMO fiber angle [16]. To measure the thickness of the VL muscle, we first marked the middle point of the distance between the lateral epicondyle of the femur and the greater trochanter of the hip. After ensuring that the lower limb was fixed, the probe with a sufficient amount of gel was placed parallel to the muscle at this point. After appearing the image of the VL muscle on the monitor, we fixed the image. The maximum distance between the muscle’s superficial and deep fascia was considered the VL muscle’s thickness [22, 23]. At this point, the angle formed by the connection of the VL muscle fibers to the deep fascia was defined as the VL fibers angle [24]. To evaluate the results more objectively, we calculated the means related to the thickness and fiber angle of VMO and VL in different contraction positions as a percentage of the total resting position. Therefore, all statistical tests of contraction positions were performed on percentage ratios.

Statistical analysis
The ICC was calculated to evaluate the reliability of all dependent variables. For evaluating the normality of data related to contractile positions, the Shapiro-Wilk test was utilized. Due to the normality of all variables, a 2-way mixed ANOVA test was used to compare the healthy and PFPS groups. Also, analysis of variance with repeated measures and the Bonferroni test were performed for pairwise comparison to compare 6 different positions in each group obtained by combining hip rotations with or without ankle dorsiflexor contraction. All analyses were performed using IBM SPSS software version 24 at a significant level of P<0.05.

Results
A total of 20 healthy individuals and 20 subjects with PFPS participated in the study, whose demographic information is shown in Table 1.


At the beginning of the study, there were no statistically significant differences between the two groups in the demographic variables. The ICC results showed that the reliability of dependent variables during maximal SLR contraction was greater than 0.95 (Table 2).


The values ​​of the VMO and VL muscle thickness and fiber angle, as well as the ratio of VMO to VL thickness at rest (baseline position) for healthy and PFPS subjects, are given in Table 3.


The results indicated that the VMO and VL muscle thickness and fiber angle and the ratio of VMO to VL thickness in PFPS subjects were significantly lower than in healthy individuals at baseline position (P=0.01). 
In the between-group comparison of contractile positions, the results showed no significant difference in any of the variables for the group factor (P>0.05, Table 4).


However, the main effect of contractile positions in both healthy and PFPS groups during the SLR maneuver was significant (P<0.05), and the results are presented below.
The independent effect of hip rotation without ankle dorsiflexion contraction on VMO thickness changes showed that hip rotation during SLR had a significant effect on VMO thickness so that the existence of external hip rotation could significantly increase this outcome in both groups compared to neutral and internal hip rotations (P=0.01). On the other hand, the evaluation of the combined hip rotation and ankle position showed that performing SLR exercise with external hip rotation and ankle dorsiflexion compared to SLR with neutral hip rotation and no ankle dorsiflexion significantly increased VMO thickness and fiber angle in both groups (P=0.01) (Figure 2).  Also, a statistically significant effect was observed regarding the independent effect of hip rotations on changes in VL thickness and fiber angle, so internal hip rotation increased these outcomes in both groups compared to the neutral hip position (P=0.01). Moreover, internal hip rotation and ankle dorsiflexor contraction significantly increased VL thickness and fiber angle compared to SLR with a neutral hip position and no ankle dorsiflexion in both groups (P=0.01) (Tables 5 and 6).




Regarding the thickness ratio of VMO to VL in different SLR positions, the addition of ankle dorsiflexor contraction to the exercise was an influential factor in this ratio, so this variable during SLR with external hip rotation and no ankle dorsiflexor contraction was significantly higher than SLR with external rotation of the hip without ankle dorsiflexion in the two groups (P=0.01). Nevertheless, no significant difference was observed in the combined effect of hip rotation and ankle position on this ratio (Figure 3).
Discussion
This study aimed to find the best position to activate VMO muscle than VL in different positions of SLR maneuver in two groups of healthy people and those with PFPS. Various techniques, such as magnetic resonance imaging, electromyography, and ultrasonography, are used to study skeletal muscle structure and function; ultrasonography, as a non-invasive, accessible, and inexpensive tool, can easily examine these outcomes in either static or dynamic positions [25]. In this study, we utilized ultrasonography to evaluate VMO and VL activation during the maximal SLR maneuver with different positions of hip rotation and ankle dorsiflexion. Ultrasonography showed high reliability in examining the structural features of VMO and VL during SLR.
VMO and VL muscle thickness and fiber angle in the PFPS group were significantly less than in healthy people at rest. This finding is consistent with the results of Giles et al. (2013) and Dong et al. (2021), reporting atrophy in the VMO and VL muscles at rest position for PFPS patients compared to healthy individuals. The reason for this claim was the presence of pain during quadriceps contraction, which led to inhibition of this muscle, resulting in atrophy in both VMO and VL muscles in these patients [26, 27].
In between-group comparison, no significant difference in the VMO and VL muscle thickness and fiber angle was observed in different positions of SLR maneuver, and the trend of changes in the two groups followed the same pattern by changing the hip rotation with or without ankle dorsiflexor contraction. These results are consistent with the findings of Lotfi et al. (2018), who reported no significant difference in the electrical activity ratio of VMO to VL during the SLR maneuver between the healthy and PFPS groups [28]. The lack of between-group difference could be young PFPS patients in the present study with no chronic condition, and the pattern of their knee muscles activity probably did not substantially change compared to healthy individuals. Also, because these patients did not have much severe pain, they could probably activate the quadriceps muscle as much as healthy people.
Based on the results, the independent position of the external hip rotation compared with the two other hip rotations led to a significant increase in the VMO muscle thickness and fiber angle. This finding confirms that reported by Sykes et al. (2003) study [9]. It may be due to the connections of the VMO muscle to the adductor magnus and the adductor longus; the fibers of these muscles are placed on the surface, thereby performing the SLR with hip external rotation by the collaboration of adductors [1]. As a result of more activity of the adductor muscles, the VMO muscle also shows more activity by increasing the values of the sonographic parameters of the present study. On the other hand, with the hip external rotation, the VMO should overcome greater gravitational force, resulting in greater torque production in the quadriceps muscle. In this regard, Mikaili et al. (2017) showed that greater force was produced in the external hip rotation compared to the internal and neutral rotations of the hip during the SLR maneuver in healthy individuals [29]. However, Livecchi et al. (2002) found that external hip rotation during SLR could not be used as a specific exercise to activate VMO in healthy individuals [12]. In previous studies, surface EMG has been utilized to evaluate the VMO and VL muscle activation in SLR maneuvers with different hip rotations. However, in the present study, ultrasonography was applied, which may be a reason for the controversy between the results of this study and some previous studies.
Adding ankle dorsiflexion to the SLR maneuvers significantly increased the VMO and VL thickness and fiber angle in healthy and PFPS groups. This finding is in line with that of Choi et al. (2014) and Cha et al. (2014) [10, 30]. From the point of view of neurophysiology and according to the overflow principle in proprioceptive neuromuscular facilitation (PNF), the contractile level of muscles can be affected by the contraction of other muscles. This energy transfer is justified by the irradiation principle, in which the torque resulting from the contraction of other muscles is transferred to weaker motor units [31]. Using the electromyographic examination of the abdominal muscles, Chon et al. (2010) showed that the addition of ankle dorsiflexion to the abdominal drawing-in maneuver increased the activity of the abdominal muscles through the irradiation [32]. The existence of a reflex relationship between the pretibial muscle and the quadriceps muscle has also been shown in the literature; for example, Kalantari et al. (2009) and Rafsanjani et al. (2017) investigated this reflex relationship, and their findings are consistent with the results of the present study [33, 34]. Moreover, the extent and intensity of this reflex vary at different positions of the knee and the hip joints. As the knee extension and hip flexion move closer to their terminal range of motion, the incidence of this reflex reaches its maximum. This reflex appears to occur more often in the SLR exercise performed at their terminal range of motion of the knee and hip. This event may explain the knee muscles’ increased thickness, fiber angle during the SLR, and ankle dorsiflexor contraction.
The results of our study also indicated that the hip internal rotation with ankle dorsiflexion during the SLR maneuver compared to other positions significantly increased in VL muscle thickness and fiber angle. Because the VL muscle is placed on the surface resulting from the hip’s internal rotation, it becomes more active during the SLR than other parts of the quadriceps. This result was also confirmed by Serrao et al. (2005). They stated that the internal rotation of the tibia increased the electrical activity of the VL muscle in isometric knee extension exercises [8].
This study evaluated the ratio of VMO to VL thickness as a measure of VMO activation. In exercise therapy in PFPS patients, the emphasis should be on specific VMO strengthening [35]. The combination of hip rotations and ankle dorsiflexion on the thickness ratio of VMO to VL indicated no significant difference between the healthy and PFPS groups. This result is consistent with the results of Pattyn et al. (2011). They showed that the ratio of the cross-sectional area of VMO muscle to VL in PFPS individuals was not significantly different compared to healthy individuals [36]. Similarly, atrophy in both VMO and VL muscles in their distal part resulted in no significant difference in the ratio of the cross-section of VMO muscle to VL in PFPS patients than in healthy individuals.

Study Limitations
One of the limitations of this study was that we just included young participants. It is suggested that future studies are conducted on older people and other musculoskeletal knee injuries. Another limitation was the immediate evaluation of exercise on sonographic parameters; thus, a high-scale design with a long-term exercise program could help find the best exercise for greater VMO activation in PFPS patients.

Conclusion
The present study indicated that by changing hip rotations with or without ankle dorsiflexion during SLR, the changes in VMO and VL thickness and fiber angle in the two groups followed the same pattern. Moreover, performing SLR in hip external rotation with ankle dorsiflexion can be recommendable for the rehabilitation of PFPS.

Ethical Considerations
Compliance with ethical guidelines
This study was approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences (Code: IR.SBMU.RETECH.REC.1400.217). The participants were informed about the study objectives and were assured that their information would remain confidential. They all signed a written consent form.

Funding
This article was extracted from the PhD thesis of Saeed Mikaili. The study was funded by Shahid Beheshti University of Medical Sciences. 

Authors' contributions
Conceptualization: Saeed Mikaili and Khosro Khademi Kalantari; Methodology, and data analysis: Minoo Khalkhali Zavieh and Alireza Akbarzadeh Baghban; Investigation: Saeed Mikaili and Mehdi Banan Khojasteh; Editing and review: Aliyeh Daryabor; Supervision: Khosro Khademi Kalantari.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors would like to thank the Vice-Chancellor for Research of Shahid Beheshti University of Medical Sciences for financial support and all participants for their cooperation.

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