# Muscle composition is not a prognostic factor for muscle strength recovery after anterior cruciate ligament surgery by hamstring tendon autograft

Maxime Pingon, Gaspard Fournier, Jobe Shatrov, Ando Radafy, Clara Bernard, Julien Gondin, Sébastien Lustig, Elvire Servien

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## To cite this version:

Maxime Pingon, Gaspard Fournier, Jobe Shatrov, Ando Radafy, Clara Bernard, et al., Muscle composition is not a prognostic factor for muscle strength recovery after anterior cruciate ligament surgery by hamstring tendon autograft. Orthopaedics & Traumatology: Surgery & Research, 2025, 111 (6), pp.104111. 10.1016/j.otsr.2024.104111. hal-04855999

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Submitted on 13 Oct 2025

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Original article

# Muscle composition is not a prognostic factor for muscle strength recovery after anterior cruciate ligament surgery by hamstring tendon autograft

Maxime Pingon<sup>a,*</sup> <sup>●</sup>, Gaspard Fournier<sup>a</sup>, Jobe Shatrov<sup>a</sup>, Ando Radafy<sup>a</sup>, Clara Bernard<sup>b</sup>, Julien Gondin<sup>b</sup>, Sébastien Lustig<sup>a,c</sup>, Elvire Servien<sup>a,d</sup>

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## ARTICLE INFO

**Keywords:**

Anterior cruciate ligament

Anterior cruciate ligament reconstruction

Muscle fibers

Immunohistochemistry

Hamstrings muscle

Return to sport

Isokinetic

## ABSTRACT

**Purpose:** For the athlete, anterior Cruciate Ligament (ACL) rupture and its surgical management are often a turning point in their career. Success and time to return to sport are essential parameters for athletes and their support staff, so it is critical to understand the prognostic factors influencing return to sport after anterior cruciate ligament reconstruction (ACLR). The aim of this study was to determine the influence of hamstring muscle composition on muscle power following ACLR with autogenous hamstring grafts.

**Methods:** 24 patients with chronic torn ACL were included at a single-center over a period of 17 months. They underwent surgical repair and during this procedure grafts were harvested from the gracilis and the semitendinosus. Muscle composition was assessed on the remaining proximal part of the *semitendinosus* muscle, which is usually discarded, by immunostaining. Muscle power was defined by comparing the strength of the operated leg and the healthy leg on an isokinetic dynamometer at 6 months according a standardized protocol after 6 months of outpatient rehabilitation. Various other intrinsic and extrinsic factors were also studied, such as body mass index (BMI), age, sex, smoking, or sport practiced, to determine factors influencing isokinetic strength test after ACLR.

**Results:** No statistical relationship was identified between muscle composition and the muscle power between the operated and healthy leg. Smoking and female gender were associated with worse muscle recovery. Age and BMI had no influence on isokinetic performance at 6 months.

**Conclusion:** Following ACLR muscle composition is not associated with difference in strength between the two legs at 6 months. Determining muscle fiber composition of the patient does not inform the rehabilitation protocol or predict muscle strength recovery. Larger series data is required to understand the influence of gender or tobacco on muscle fiber characteristic.

**Level of Evidence:** I; Prospective prognostic study.

## 1. Introduction

For an athlete, anterior cruciate ligament (ACL) tear can be a turning point in their career. A rapid return to sport (RTS) is often the primary objective for the player. A variety of factors have been shown to effect the success and time to RTS [1].

On average, more than 80% of patients return to physical activity, two-thirds of whom return to their pre-injury level of sport, and half of whom return to competitive sport after surgery [2-4]. Patients returning

to sport who fail to meet objective criteria have been shown to be up to 4 times more likely to re-injure their ACL reconstruction (ACLR) or injure the contralateral side [5]. Typically, RTS testing takes place from 6 months after surgery, and consists of a battery of physical tests and functional questionnaires [6-11]. A commonly used criteria for RTS is the symmetry of force production of the flexors and extensors of the lower limb [12-14]. To date, there are no intrinsic factors, other than age and gender, that predict RTS after ACLR [15]. The study of muscle typology and its influence on performance is still poorly evaluated but is

<footnote><sup>a</sup> Orthopaedics Surgery and Sports Medicine Department, FIFA Medical Center of Excellence, Croix-Rousse Hospital, Lyon University Hospital, Lyon, France</footnote>

<footnote><sup>b</sup> Institut NeuroMyoGène, Unité Physiopathologie et Génétique du Neurone et du Muscle, Université Claude Bernard Lyon 1, CNRS UMR 5261, Inserm U1315, Lyon, France</footnote>

<footnote><sup>c</sup> University of Lyon, Claude Bernard Lyon 1 University, IFSTTAR, LBMC UMR T9406, F69622, Lyon, France</footnote>

<footnote><sup>d</sup> LIBM - EA 7424, Interuniversity Laboratory of Biology of Mobility, Claude Bernard Lyon 1 University, Lyon, France</footnote>

<footnote>* Corresponding author.
E-mail address: maxime.pingon@chu-lyon.fr (M. Pingon).</footnote>
receiving increasing scientific interest [16–18].

Three types of muscle fibers have been described in humans, I, IIA and IIX [19–22]. Each type is characterized according to its myosin heavy chain (MHC) isoform composition [22,23]. This defines the metabolic and clinical characteristics of the fibers [19,24]. Type I fibers have high oxidative and glycolytic activity, providing a higher endurance capacity. In contrast, type II fibers, a group that includes type IIA and IIX fibers in humans, are described as fast fibers and produce energy through the anaerobic pathway [16,17,25]. The inter-individual variability in this composition is a source of very different performance and resistance to exercise [26]. A higher proportion of fast fibers produces both more power and speed [16,27]. Histologically, the quadriceps or hamstring muscles have a composition rich in type II fibers [20,28–30].

Immobilization, following injury or surgery, results in a change in muscle composition from slow to fast [31,32]. During immobilization, these so-called fast fibers tend to atrophy more significantly than type I fibers, by 13 ± 4% and 7 ± 3%, respectively [33], especially type IIA fibers [9,34]. This change in composition also occurs after ACLR [35]. Conversely, training appears to increase the proportion of type IIA fibers at the expense of type IIX fibers without any change in the quantity or proportion of type I fibers [36]. Finally, during a training or exhaustion test, the maximum strength of patients with a muscle composition rich in fast fibers decreases more significantly and the recovery time of maximum capacities is higher [17]. Subjects with a more rapid fiber typology may therefore have a longer time to return to their previous level.

Given the characteristics of the different types of muscle fibers (tendency to atrophy, ability to regenerate, force production, endurance resistance, etc.), muscle composition may predict recovery capacity and RTS following ACLR.

The primary aim of this study was to determine the relationship between hamstring muscle typology and isokinetic muscle power and strength 6 months after ACLR. Secondary aim was to investigate the influence of intrinsic factors (age, gender, body mass index {BMI}, smoking, sport) on muscle power and strength.

# 2. Materials and methods

## 2.1. Population

Forty-two patients were prospectively included in this study between November 2019 and March 2021. Inclusion criteria were: total rupture of the ACL with functional instability, indication for ACLR with semitendinosus and gracilis tendons (STG), age between 18 and 50 years. Patients were excluded if they had a history of hamstring injury, a neuromuscular pathology or had received treatment with corticosteroids in during the study period. All eligible patients admitted to the medical-surgical center received information about the study, and all agreed to take part in the follow-up.

Functional instability was defined by the presence of a chronic rupture of the ACL confirmed by MRI, associated with a clinical rotatory shift and a soft-stop Lachman test.

Eight patients had lateral meniscus damage, 8 patients had medial meniscus damage, and only one patient had damage to both menisci. Treatment of these lesions was based on current recommendations [6, 37].

## 2.2. ACLR and muscle harvesting

The ACL reconstruction technique was based on the so-called “all-inside” technique, using shallow bone tunnels for maximum cortical bone preservation. Hamstring tendons, such as the *semitendinosus*, are prepared to form a suitable graft. Cortical fixation buttons are used to ensure reliable anchoring in the tibia and femur. The approach minimizes surgical trauma through limited incisions and aims to optimize functional recovery. During ACLR with hamstring harvesting, a remnant

of the proximal portion of the semitendinosus muscle is usually discarded. This remnant was isolated using sharp dissection and preserved in ice before transport and preparation for muscle composition study. All surgical procedures were performed by the same surgeon.

## 2.3. Muscle analysis

### 2.3.1. Myosin heavy chain immunostaining

The protocol used was adapted from a murine protocol (Rao et al., 2019 [38], Wang et al., 2017 [39]). Muscle biopsies were directly frozen in isopentane placed in liquid-nitrogen and kept at -80 °C until use. Cryosections (10 µm) were prepared.

Cryosections were incubated with primary antibodies overnight at 4 °C, washed with phosphate-buffered saline (PBS) and further incubated with secondary antibody for 1 h at 37 °C. The following antibodies were used: anti-MHC-I (mouse, BA-D5, 1/100), anti-MHC-IIA (mouse, SC-71, 1/100) and anti-MHC-IIX (mouse, 6H1, 1/100) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City) and anti-laminin (rabbit, L9393, 1/200, Sigma-Aldrich, St. Louis). Secondary antibodies (Jackson Immunoresearch) were Alexa fluor 405 IgG2b (anti-mouse MyHC-I, 1/200), Alexa Fluor 488 IgG1 (anti-mouse MyHC-IIA, 1/200), Alexa Fluor 546 IgM (anti-mouse MyHC-IIX, 1/200) and Cy-5 (anti-rabbit, 1/200). Slides were washed with PBS and mounted in Fluoromount-G.

### 2.3.2. Quantification

Four images were randomly recorded on each cryosection using a Zeiss Imager Z1 microscope at 20x magnification connected to a CoolSNAP MYO camera. Using the antibody combination described, MHC-I cells were labeled blue, MHC-IIA green, MHC-IIX red, and laminin was artificially labeled white (Fig. 1). The number of fibers was determined with ImageJ software [40] on the Fiji platform [41]. All images were acquired and analyzed in a blinded fashion (364 fibers per patient on average).

## 2.4. Rehabilitation

All patients underwent a post-operative rehabilitation program. This was supervised by a physiotherapist on an outpatient basis, according to an established program handed out in the form of a schedule with milestones. The aim of the first 3 weeks was to limit arthrogenic motor inhibition [42] and remove flessum. Thereafter, the progressive increase in workload depended on the patient's progress, with the aim of returning to running at 3 months after surgery. The physiotherapist was blinded to the muscle typology.

## 2.5. Isokinetic muscle testing protocol

All isokinetic testing was supervised by a sport physician at 6 months post ACLR. The operator was blind of the Myosin heavy chain immunostaining results before testing.

### 2.5.1. Warm-up

A preliminary warm-up was performed on a cycloergometer. The patient's goal was a pedaling speed of 60 rotations per minute. The resistance of the device was adjusted at the patient's discretion with the aim of reaching a power of 1 Watt.kg⁻¹ during the last minute. A second specific warm-up on the isokinetic tool was then performed so that the patient could become familiar with the machine and control for a learning effect.

### 2.5.2. Setup

The patients were installed and secured on a PHYSIOMED Con-Trex isokinetic dynamometer in a seated position at 85 ° of hip flexion according to the manufacturer's recommendations (0 ° corresponding to full extension). The amplitude of the dynamometer arm was calibrated
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Illustration of a muscle section after immunostaining seen under an epifluorescence microscope before and after counting. Fiber staining. Blue: type I fiber, Green: type IIA fiber, Red: type IIX fiber, White: laminin.</figcaption></figure>

before fixing the patient's leg for an angular stroke of 85° between 15° and 100° (0° corresponding to full extension). Calibration and gravity correction was performed (the machine's computerized correction considers the weight of the patient's leg in order to exclude it during the force recordings) [14,43,44].

### 2.5.3. Protocol

The isokinetic analysis started with the healthy leg (Table 1) with a series of 3 maximum concentric contractions in extension and flexion at 60°.s⁻¹ followed by a series of 4 maximum concentric contractions in extension and flexion at 240°.s⁻¹ and finally a series of 2 maximum eccentric contractions testing only the flexors at 30°.s⁻¹. The patients were instructed to exert as much force as possible during each contraction and received standardized oral motivation from the operator present throughout the test. All the isokinetic evaluations were performed on the same apparatus, under the same examination conditions, by the same operator.

### 2.5.4. Measure and data analysis

The patients' maximal force was measured automatically by the dynamometer, which provided the best peak torque achieved on the series at a given speed as well as a curve representing the power applied at each degree of the movement.

The difference between the two limbs was then calculated as a percentage of force recovery compared to the healthy contralateral side [14,43] according to the following formula:

$$ \frac{\text{peak torque of the healthy leg} - \text{peak torque of the operated leg}}{\text{peak torque of the healthy leg}} \times 100 $$

The mixed ratio, studying the ratio between the concentric force of the extensors and the eccentric force of the thigh flexors, was calculated and presented in absolute values. The study of extensor and flexor ratios is predictive of the risk of graft rupture as well as hamstring injury [5,7].

### 2.6. Statistical analysis

Statistical analyses were performed using R software with the XQuartz tool and with the online software pvalue (Medistica. pvalue.io, a Graphic User Interface to the R statistical analysis software for

<table><caption>**Table 1**

Isokinetic evaluation protocol for each leg.</caption><thead><tr><th></th><th>isokinetic mode</th><th>Angular Speed (°/sec)</th><th>N° contraction</th></tr></thead><tbody><tr><td rowspan="2">Warm Up</td><td>concentric</td><td>60</td><td>3</td></tr><tr><td>concentric</td><td>240</td><td>4</td></tr><tr><td rowspan="3">Test</td><td>concentric</td><td>60</td><td>3</td></tr><tr><td>concentric</td><td>240</td><td>4</td></tr><tr><td>eccentric</td><td>30</td><td>2</td></tr></tbody></table>

scientific medical publications. 2020. Available at: https://www.pvalue.io/fr, n.d.). Data are presented as mean ± standard deviation unless otherwise noted.

Descriptive analyses were first performed for each variable of interest. For the analysis of means, a univariate t-test was used. In the univariate t-test, the null hypothesis was defined as muscle composition equal to 50% and muscular strength discrepancy equal to 0%. A calculation of Spearman's correlation coefficient was performed during the correlation analyses.

A value of p < 0.05 was considered significant for each analysis.

### 2.7. Ethics

The protocol for this study was submitted and accepted by an ethics committee (N°19-52).

## 3. Results

Forty-two patients were initially included. Eighteen subjects had to be excluded. Eleven patients were unable to perform the muscle testing, either because of insufficient range of motion or pain or because they were lost to follow-up, and 7 muscle samples could not be studied by immunostaining due to insufficient sample quality (destruction during transport, thawing due to material damage, or damage to the muscle during sampling). This yielded a total of twenty-four muscle samples available (Fig. 2) for final analysis at 6 months (p = 0.9) after surgery (Fig. 2). Patient characteristics are shown in Table 2.

The mean time between surgery and testing was 6 months and 11 days and the mean muscle deficit was 17.2% during concentric evaluation at 240°.s⁻¹ (p < 0.0001; CI [12.9; 30.4]), 21.7% at 60°.s⁻¹ (p < 0.001; CI [54.3; 62.4]) and 28.7% eccentric at 30°.s⁻¹ (p < 0.0001; CI [21.4; 36.0]). Hamstring muscles were predominantly (58.4%) composed of type II fibers (p < 0.001; CI [54.3; 62.4]) (Fig. 3).

### 3.1. Primary outcome

No statistically significant correlation was found between the percentage of fibers (I, II, IIA, IIX) and isokinetic indices, nor between the number of fibers (I, II, IIA, IIX) and isokinetic indices (Fig. 4).

### 3.2. Secondary outcomes

#### 3.2.1. Analysis by gender

Men developed statistically more concentric strength relative to body weight than women: 1.09 vs. 0.755 Nm.kg⁻¹ (p = 0.049). However, there was no significant difference on the other isokinetic indices at 6 months post-op (Table 3).
<figure><img src="image_6.png" /><figcaption>Fig. 2. Flow Chart.</figcaption></figure>

<table><caption>**Table 2**

Baseline characteristics of patients.</caption><thead><tr><th rowspan="2">Characteristics</th><th colspan="2">Patients (n = 24)</th></tr><tr><th>mean (sd)</th><th>range</th></tr></thead><tbody><tr><td>Age (Year)</td><td>32.5 (10.3)</td><td>19–49</td></tr><tr><td>BMI (kg/m<sup>-2</sup>)</td><td>25.3 (5.82)</td><td>20.2–48.5</td></tr><tr><td>Delay (month)</td><td>6.03 (0.918)</td><td>4.30–8.20</td></tr><tr><td>Muscle Composition</td><td></td><td></td></tr><tr><td>Number of Type I Fiber</td><td>147 (55.4)</td><td>67–287</td></tr><tr><td>Number of Type II Fiber</td><td>218 (116)</td><td>83–594</td></tr><tr><td>Number of Type IIA Fiber</td><td>101 (56.5)</td><td>42–302</td></tr><tr><td>Number of Type IIX Fiber</td><td>117 (65.7)</td><td>38–292</td></tr><tr><td>Total</td><td>365 (157)</td><td>170–881</td></tr><tr><td>Percentage Type I Fiber</td><td>41.6 % (9.6)</td><td>20.9–61</td></tr><tr><td>Percentage Type II Fiber</td><td>58.4 % (9.6)</td><td>39–79.1</td></tr><tr><td>Percentage Type IIA Fiber</td><td>27.5 % (6.6)</td><td>16.1–37.6</td></tr><tr><td>Percentage Type IIX Fiber</td><td>30.9 % (7.4)</td><td>16.1–46.2</td></tr><tr><td>Isokinetic</td><td></td><td></td></tr><tr><td>Maximal concentric Strength 60/kg</td><td>0.9 (0.4)</td><td>0.150–1.65</td></tr><tr><td>Concentric deficit 240 (H/H)</td><td>17.2 % (13.9)</td><td>(–)3–55.8</td></tr><tr><td>Concentric deficit 60 (H/H)</td><td>21.7 % (20.6)</td><td>(–)15.8–71</td></tr><tr><td>Excentric deficit 30 (H/H)</td><td>28.7 % (17.3)</td><td>2.7–67</td></tr><tr><td>Mixed Ecc 30/Conc 240</td><td>1.38 (0.8)</td><td>0.66–4.49</td></tr></tbody></table>

### 3.2.2. Analysis by smoking status

In our population, 8 patients reported smoking (33%). Smoking status was determined according to whether or not the patient considered him/herself a smoker at the time of the medical interview. Muscle composition was not influenced by patient smoking (Table 4). In contrast, we showed that the patient's active smoking was significantly correlated with a greater differential in hamstring eccentric strength. Smoking patients had on average a 41.4% deficit with the index leg versus 22.3% for non-smoking patients (p = 0.016). No significant differences were found for the other indices, but a clear trend towards

delayed recovery in the smoking patients emerged.

### 3.2.3. Analysis by age

No significant correlation between age and muscle composition or isokinetic indices was identified. Subgroup analysis (Young: less than 25 years; Middle: between 25 and 35 years; Old: more than 35 years) did not find a correlation (Table 5).

### 3.2.4. Analysis by BMI

BMI did not correlate statistically with muscle composition, nor with isokinetic indices. No correlation could be found in the subgroup analysis (Healthy [<25] vs. Overweight [>25] and Healthy [<25] vs. Overweight [>25] vs. Obese [>30]) (Table 5).

## 4. Discussion

This study assessed hamstring muscle composition and determined its influence on isokinetic performance at 6 months after ACLR with hamstring autograft. The most important results of the study are that no significant correlation was found between muscle typology and muscle strength deficit at six months after ACLR. We also reveal a balanced composition of semitendinosus muscle typology, composed of 41.6%, type I fibers, 27.5% type IIA and 30.9% type IIX, i.e., 58.4% had type II typology.

To our knowledge, the typology of the human semitendinosus muscle was recently assessed using immunostaining, which is the reference technique for muscle histological analysis [39,45]. This recent investigation was in line with the seminal work of Garret et al. on cadaveric samples [20] which showed that the distal portion of the semitendinosus was composed mainly of type II fibers with a proportion ranging from 50 to 69.5%. Other studies have shown more contrasting results with proportions of 47.1 ± 9.1% of type I fibers when studying the biceps femoris muscle [29]. These studies used electrophoresis to establish muscle typology. Garret et al. presented similar results when studying other thigh muscles, but other researchers have looked at this and shown that the proportion of type II fibers is even greater in the quadriceps with only
<figure><img src="image_7.png" /><figcaption>Fig. 3. Graphic representation of the hamstring muscle typology.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 4. Influence of muscle composition on different isokinetic indices.</figcaption></figure>
<table><caption>**Table 3**
Influence of sex on muscle composition and testing.</caption><thead><tr><th>Sex</th><th>Male (n = 13)</th><th>Female (n = 11)</th><th>p</th></tr></thead><tbody><tr><td>Age (Year)</td><td>29.6 (±7.6)</td><td>36.0 (±12.2)</td><td>0.19</td></tr><tr><td>BMI (kg.m<sup>-2</sup>)</td><td>27.0 (±7.1)</td><td>23.3 (±3.1)</td><td>0.064</td></tr><tr><td>Delay (month)</td><td>6.3 (±1.1)</td><td>5.7 (±0.5)</td><td>0.15</td></tr><tr><td>Muscle Composition</td><td></td><td></td><td></td></tr><tr><td>Number of Type I Fiber</td><td>116 (±45)</td><td>183 (±45)</td><td><strong>&lt;0.01</strong></td></tr><tr><td>Number of Type II Fiber</td><td>179 (±87)</td><td>264 (±132)</td><td>0.055</td></tr><tr><td>Number of Type IIA Fiber</td><td>87. (±38)</td><td>117 (±71)</td><td>0.3</td></tr><tr><td>Number of Type IIX Fiber</td><td>92 (±56)</td><td>147 (±66)</td><td><strong>0.016</strong></td></tr><tr><td>Total</td><td>295 (±103)</td><td>447 (±173)</td><td><strong>0.019</strong></td></tr><tr><td>Percentage Type I Fiber</td><td>40.7% (±11.9)</td><td>42.7% (±6.3)</td><td>0.78</td></tr><tr><td>Percentage Type II Fiber</td><td>59.3% (±11.9)</td><td>57.3% (±6.3)</td><td>0.78</td></tr><tr><td>Percentage Type IIA Fiber</td><td>29.7% (±7.1)</td><td>24.9% (±5.3)</td><td>0.072</td></tr><tr><td>Percentage Type IIX Fiber</td><td>29.6% (±8.6)</td><td>32.4% (±5.8)</td><td>0.25</td></tr><tr><td>Isokinetic</td><td></td><td></td><td></td></tr><tr><td>Maximal concentric Strength 60/ kg</td><td>1.1 (±0.36)</td><td>0.76 (±0.34)</td><td><strong>0.049</strong></td></tr><tr><td>Concentric deficit 240 (H/H)</td><td>13.7% (±11.9)</td><td>21.3% (±15.6)</td><td>0.39</td></tr><tr><td>Concentric deficit 60 (H/H)</td><td>15.4% (±16)</td><td>29% (±23.6)</td><td>0.23</td></tr><tr><td>Excentric deficit 30 (H/H)</td><td>23.3% (±14.6)</td><td>35.1% (±18.7)</td><td>0.16</td></tr><tr><td>Mixed Ecc 30/Conc 240</td><td>1.5 (±1.0)</td><td>1.3 (±0.4)</td><td>0.69</td></tr></tbody></table>

<table><caption>**Table 4**
Influence of smoking on muscle composition and testing.</caption><thead><tr><th>Smoker</th><th>No (n = 16)</th><th>Yes (n = 8)</th><th>p</th></tr></thead><tbody><tr><td>Muscle Composition</td><td></td><td></td><td></td></tr><tr><td>Number of Type I Fiber</td><td>140 (±59.5)</td><td>161 (±46.4)</td><td>0.22</td></tr><tr><td>Number of Type II Fiber</td><td>204 (±120)</td><td>247 (±107)</td><td>0.24</td></tr><tr><td>Number of Type IIA Fiber</td><td>102 (±61.9)</td><td>99.9 (±47.6)</td><td>0.88</td></tr><tr><td>Number of Type IIX Fiber</td><td>102 (±61.2)</td><td>147 (±67.8)</td><td>0.12</td></tr><tr><td>Total</td><td>343 (±169)</td><td>408 (±127)</td><td>0.16</td></tr><tr><td>Percentage Type I Fiber</td><td>41.8% (±9.8)</td><td>41.2% (±9.8)</td><td>0.93</td></tr><tr><td>Percentage Type II Fiber</td><td>58.2% (±9.8)</td><td>58.8% (±9.8)</td><td>0.93</td></tr><tr><td>Percentage Type IIA Fiber</td><td>29% (±6.2)</td><td>24.4% (±6.9)</td><td>0.14</td></tr><tr><td>Percentage Type IIX Fiber</td><td>29.1% (±6.4)</td><td>34.4% (±8.6)</td><td>0.12</td></tr><tr><td>Isokinetic</td><td></td><td></td><td></td></tr><tr><td>Maximal concentric Strength 60/ kg</td><td>0.98 (±0.40)</td><td>0.86 (±0.30)</td><td>0.56</td></tr><tr><td>Concentric deficit 240 (H/H)</td><td>15.6% (±10.6)</td><td>20.3% (±19.5)</td><td>0.61</td></tr><tr><td>Concentric deficit 60 (H/H)</td><td>18.5% (±19.8)</td><td>28.5% (±21.8)</td><td>0.32</td></tr><tr><td>Excentric deficit 30 (H/H)</td><td>22.3% (±14.1)</td><td>41.4% (±16.6)</td><td><strong>0.016</strong></td></tr><tr><td>Mixed Ecc 30/Conc 240</td><td>1.42 (±0.9)</td><td>1.31 (±0.6)</td><td>0.49</td></tr></tbody></table>

36% type I fibers in the *vastus lateralis* [16]. There is therefore a very large inter- and intra-individual typological variability whose influence on performance is not well understood.

The evaluation of our cohort did not reveal any statistical correlation between muscle composition and isokinetic muscle strength. Muscle composition that evaluates the proportion of myosin heavy chain may not be sufficient to define the performance of an athlete in the field [21]. Various cellular or extracellular elements are studied and described in the literature [25]. For example, it was recently demonstrated that the quantity of satellite cells, muscle stem cells, is directly correlated to the regenerative capacity of skeletal muscle [46]. The study of overall muscle cell composition may require assessment of their role in muscle

strength recovery after ACL [47].

In vitro, force production capabilities are widely evaluated, but it is still difficult to assess this performance in real-world exercise [24,26]. As described by Evangelidis et al. [29], there is no strong evidence of the influence of composition on muscle performance in real-life sporting conditions [16]. However, other studies have shown a positive correlation between the proportion of type II fibers and maximal strength/-velocity [48]. Regardless of fiber type, strength and velocity production are dependent on their level of training [49]. Exercise has little effect on typology despite improved performance. This data provides an element of response to the lack of correlation between typology and muscle recovery. This reinforces the idea that the rehabilitation protocol and the follow-up of the patients are essential for the return to sport.

Although an expected difference was observed in the peak power developed on an isokinetic machine between men and women, no significant difference in the power deficit between the 2 legs was observed according to gender. Males showed a smaller difference in strength between the two lower limbs than women. During the evaluation at 60°, s⁻¹, males had a deficit of 15% compared to 29% for the females in the current study. The literature on gender differences and muscle strength remains limited. However in the medium- and long-term follow-up, males are more likely to return to their previous level of sport and have better functional scores than women [2,50]. Ahldén et al. had studied a cohort of 141 men and 103 women who underwent STG ACLR in 2012. The population showed comparable recovery of functional scores but differences on the only functional test used (One-Leg-Hop test) [51]. Male gender appears to be a prognostic factor for favorable outcome after ACLR [15,52] without the difference in typology being a confounding factor. The etiology for these observed differences will require further research.

The impact of smoking in patients was also evaluated. Smoking is widely recognized as a poor prognostic factor following ACLR. In the postoperative phase, the risk of thromboembolic disease or surgical site infection is higher in smokers. In the medium term, the rate of repeat rupture is increased and functional scores are worse than in patients who are non-smokers [53]. Chronic smoking is a factor in poor functional recovery in the medium [54] and long term with persistent pain, even at 10 years after surgery [50]. In the current study this was manifested by a greater strength deficit without any difference in muscle composition. The findings of the current study add to the broader body of evidence that highlight the importance of smoking cessation, starting in the preoperative phase.

The return to sporting activity remains the main objective for many patients following ACLR. Knowledge of favorable or unfavorable prognostic factors would initially make it possible to modulate the surgical indication. These factors could also make it possible to adapt rehabilitation to each patient, by setting different objectives according to the profile of favorable or unfavorable evolution. Further investigations are needed to define these factors.

## 5. Limitations

The main limitation of this work is the proportion of patients lost to follow-up in the study population. This number combined with the number of excluded patients exceeded expectations. This led to a limited sample size and could have resulted in a Type II error. It is important to note that the follow-up period included all 3 of the Covid 19 pandemic containment regimes, the adverse effects of which on access to scheduled care are widely recognized [55]. Patients received very heterogeneous rehabilitation on an ambulatory basis, which may in part explain the low level of muscle recovery and the large number of lost to follow-up. There is therefore a follow-up and evaluation bias. However, it is quite common in this type of study to have a large proportion of lost to follow-up [51]. It would be interesting to continue this study to include more patients and hope to obtain significant results.
<table><caption>**Table 5**
Influence of Age and BMI on muscle composition and testing.</caption><thead><tr><th rowspan="2">Variable<br>Group</th><th colspan="3">Age</th><th rowspan="2">p</th><th colspan="3">BMI</th><th rowspan="2">p</th></tr><tr><th>Young<br>n = 7</th><th>Middle<br>n = 8</th><th>Old<br>n = 9</th><th>Healthy<br>n = 15</th><th>Overweight<br>n = 6</th><th>Obese<br>n = 3</th></tr></thead><tbody><tr><td><strong>Muscle Composition</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Number of Type I Fiber</td><td>145 (±49.5)</td><td>124 (±51.3)</td><td>168 (±60.2)</td><td>0.31</td><td>166 (±55.4)</td><td>118 (±40.9)</td><td>108 (±45.6)</td><td>0.17</td></tr><tr><td>Number of Type II Fiber</td><td>223 (±95.4)</td><td>153 (±51.4)</td><td>272 (±148)</td><td>0.08</td><td>252 (±127)</td><td>156 (±70.3)</td><td>174 (±72.8)</td><td>0.14</td></tr><tr><td>Number of Type IIA Fiber</td><td>107 (±45.1)</td><td>73.6 (±20.9)</td><td>121 (±77.7)</td><td>0.28</td><td>116 (±64.7)</td><td>73.0 (±27.5)</td><td>81.3 (±27.4)</td><td>0.19</td></tr><tr><td>Number of Type IIX Fiber</td><td>116 (±57.8)</td><td>79.8 (±39.1)</td><td>151 (±76.9)</td><td>0.054</td><td>135 (±66.4)</td><td>83.0 (±61.8)</td><td>92.3 (±48.1)</td><td>0.077</td></tr><tr><td>Total</td><td>369 (±123)</td><td>277 (±96.8)</td><td>440 (±192)</td><td>0.077</td><td>418 (±166)</td><td>274 (±106)</td><td>281 (±67.0)</td><td>0.084</td></tr><tr><td><strong>Isokinetic</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Percentage Type I Fiber</td><td>40.6 % (±12.8)</td><td>44.2 % (±7.4)</td><td>40.1 % (±9.2)</td><td>0.51</td><td>41.2 % (±9.9)</td><td>43.7 % (±6.4)</td><td>39.4 % (±15.5)</td><td>0.75</td></tr><tr><td>Percentage Type II Fiber</td><td>59.4 % (±12.8)</td><td>55.8 % (±7.4)</td><td>59.9 % (±9.2)</td><td>0.51</td><td>58.8 % (±9.9)</td><td>56.3 % (±6.4)</td><td>60.6 % (±15.5)</td><td>0.75</td></tr><tr><td>Percentage Type IIA Fiber</td><td>28.7 % (±8.4)</td><td>27.8 % (±6.3)</td><td>26.3 % (±6)</td><td>0.67</td><td>27.1 % (±6.8)</td><td>27.6 % (±6.8)</td><td>29.3 % (±7.6)</td><td>0.84</td></tr><tr><td>Percentage Type IIX Fiber</td><td>30.7 % (±9)</td><td>28 % (±5.50)</td><td>33.5 % (±7.5)</td><td>0.41</td><td>31.6 % (±5.7)</td><td>28.7 % (±10.2)</td><td>31.4 % (±11.2)</td><td>0.55</td></tr><tr><td>Maximal concentric Strength 60/kg</td><td>0.989 (±0.5)</td><td>1.04 (±0.3)</td><td>0.806 (±0.4)</td><td>0.45</td><td>0.953 (±0.4)</td><td>1.10 (±0.4)</td><td>0.543 (±0.2)</td><td>0.084</td></tr><tr><td>Concentric deficit 240 (H/H)</td><td>19.8 % (±19.2)</td><td>15.4 % (±12.3)</td><td>16.7 % (±11.9)</td><td>0.99</td><td>18.9 % (±14.9)</td><td>9.35 % (±12.3)</td><td>24.3 % (±5.01)</td><td>0.18</td></tr><tr><td>Concentric deficit 60 (H/H)</td><td>22.4 % (±24.3)</td><td>17.3 % (±19.9)</td><td>24.9 % (±20.0)</td><td>0.85</td><td>22.6 % (±23.2)</td><td>14.2 % (±16.9)</td><td>32 % (±7.4)</td><td>0.34</td></tr><tr><td>Excentric deficit 30 (H/H)</td><td>27.6 % (±23.6)</td><td>25.4 % (±13.4)</td><td>32.4 % (±16.0)</td><td>0.69</td><td>27.3 % (±20)</td><td>24.9 % (±10.1)</td><td>43.3 % (±3.28)</td><td>0.12</td></tr><tr><td>Mixed Ecc 30/Conc 240</td><td>1.6 (±1.3)</td><td>1.0 (±0.2)</td><td>1.5 (±0.5)</td><td>0.22</td><td>1.3 (±0.5)</td><td>1.2 (±0.3)</td><td>2.1 (±2.1)</td><td>0.93</td></tr></tbody></table>

# 6. Conclusions

Following ACLR muscle composition is not associated with difference in strength between the two legs at six months. Determining muscle fiber composition of the patient does not inform on the rehabilitation protocol needed or predict a faster return to sport.

# CRediT authorship contribution statement

MP: Study Design, patient inclusion, immunostaining, data collection, statistical analysis, literature review and manuscript writing.

GF: Manuscript editing.

JS: English proofreading & manuscript editing.

AR: Study design, patient inclusion, isokinetic testing and supervision.

CB: immunostaining and data collection.

JG: Study design, literature review and manuscript editing.

SL: Study design and supervision.

ES: Study design, supervision, literature review and manuscript editing.

All Authors read and approved the final manuscript.

# Ethical approval

Ethics committee of the Hospices Civils de Lyon N°19-52. Informed consent: Yes.

# Declaration of Generative AI and AI-assisted technologies in the writing process

No use of any generative AI or AI-assisted technologies to write this manuscript.

# Funding

FIFA.

# Declaration of competing interest

SL: Corin Institutionnal research Support, Amplitude Institutionnal research Support, Stryker Consultant ES: Corin Institutionnal research

Support/Consultant, Smith & Nephew Consultant

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