Comparison of Proprioception Recovery Following Anterior Cruciate Ligament Reconstruction Using an Artificial Graft Versus an Autograft

doi:10.21203/rs.3.rs-1628184/v1

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Research Article

Comparison of Proprioception Recovery Following Anterior Cruciate Ligament Reconstruction Using an Artificial Graft Versus an Autograft

https://doi.org/10.21203/rs.3.rs-1628184/v1

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Background

To compare proprioception recovery after anterior cruciate ligament reconstruction (ACLR) with an autograft (hamstring tendon) versus the artificial Ligament Advanced Reinforcement System (LARS).

Material and Methods

Forty patients (9 women, 31 men) with anterior cruciate ligament (ACL) rupture were enrolled in this prospective study. Patients were randomized to two groups, ACLR using a hamstring autograft or artificial LARS, with similar patient numbers per group. Proprioception was assessed with joint position sense (JPS) at 45° and 75° flexions, with the contralateral healthy knee as a control.

Results

Proprioception loss as measured by JPS deficit in both groups 3 months after ACLR (e.g., at 45° flexion, 2.81 ± 1.41 vs. 2.16 ± 0.85) was significantly higher than that at 12 months (1.87 ± 0.96 vs. 1.65 ± 0.73) (P < 0.05). However, no significant difference in proprioception loss was detected between the LARS and autograft groups at either 3 or 12 months after ACLR. Analyzing JPS deficit data showed a statistically significant difference between the groups at 3 months, but not at 12 months, after ACLR. Patients receiving the graft within 1 year of injury had a lower proprioception deficit than those receiving the graft more than 1 year after injury (e.g., at 45° flexion, 1.76 ± 0.72 vs. 3.15 ± 1.05). No complications were associated with either ACLR method.

Conclusion

ACLR with an autograft or LARS artificial graft rupture is similarly safe and effective for recovering knee proprioception, which may be primarily related to compensation by the articular capsule, muscle, and other soft tissue.

ACL reconstruction

proprioception

artificial ligament

LARS

sports medicinek

Anterior cruciate ligament (ACL) injury is one of the most common and devastating injuries of the lower extremity and a main cause of recurrent knee instability^[1]. The ACL is necessary for static and dynamic stability of the knee joint ^{[2, 3]}. The ligament’s main role in knee joint stability is to prevent excessive anterior translation of the tibia in relation to the femur and to help trigger the “screw-home” mechanism, which occurs during femoral and tibial rotation into full knee extension^[4]. Rupture of the ACL contributes to progressive functional instability and disability, which may result in secondary damage to other structures, such as meniscal tears and articular cartilage degeneration ^[5]. In addition, the ACL is thought to play a significant role in dynamic knee stability by affecting proprioception ^{[4, 6]}.

Proprioception is the synthesis of the sensory modality of joint movement, joint position, and tactile sense encompassing a joint, whether conscious or unconscious ^[7]. Proprioception is the effect of afferent nerve impulses from muscles, tendons, and joints that control reflexes and muscle tone ^[8]. The ACL not only provides mechanical stabilization but also contributes to proprioceptive functions, by means of the various mechanoreceptors within its structure, and at the same time, the ACL can detect changes in tension, speed, acceleration, direction of movement, and the position of the knee joint ^{[9, 10]}. Some specific ligament mechanoreceptors are found within the ACL, including the Ruffini corpuscles, Paccini corpuscles, and Golgi tendon organs, as well as a smaller number of free nerve endings that are important for proprioception ^{[11, 12]}. The proprioceptive and neuromuscular control of patients with ACL deficiency is diminished. The decrease of sensory information after ACL injury alters the afferent information to the central nervous system, influencing sensitivity, impairing the ability to detect motion, and inhibiting muscle motor neurons that surround the joint, changing the dynamic stability of the knee ^{[13, 14]}.

Proprioception is commonly assessed either with joint position sense (JPS), which is the ability to reproduce the target joint position actively or passively, or with threshold to detection of passive motion (TDPM), which is the ability to detect the initiation of passive joint movement ^[15]. JPS can determine where a particular body part is in space exactly by measuring the degree of angle deviation from a starting position. Muscle spindles, skin mechanoreceptors, and articular structures are likely involved in JPS ^[16–18].

There are three primary graft options for ACL reconstruction (ACLR): autograft, allograft, and synthetic graft ^[19]. Despite a vast amount of research, debate focused on the clinical outcomes of applying different grafts in ACLR continues. Because the hamstring tendon is the most common autograft used for ACLR, it was selected for use in the present study. The Ligament Augmentation and Reconstruction System (LARS) made of polyethylene terephthalate longitudinal fibers held together by transverse knitted fibers is a synthetic ligament designed specifically to overcome the failures of previous synthetic transplants and the frequently developed synovitis. Thus, LARS was selected as the synthetic graft in the present study.

Few published studies have compared knee proprioception after ACLR with an autograft vs. LARS. Thus, the goals of the present study were to assess whether proprioception improves after ACLR using LARS and to compare knee proprioception following ACLR with this synthetic graft versus the hamstring tendon autograph.

Patient selection and randomization

Patients with ACL rupture were recruited for the present study from September 2018 to February 2021. ACL rupture was diagnosed based on medical history, physical examination (positive Lachman, anterior drawer, or pivot shift tests), and magnetic resonance imaging results. The screening process is shown in Fig. 1. The criteria for selecting patients were unilateral ACL rupture as well as patient motivation and cooperation, with or without degree I/II meniscus injury, regardless of gender or profession. Standard exclusion criteria were applied and included patients who had any of the following: combined ligament injury; radiological visible degenerative changes; contralateral knee ligament injury; articular cartilage injuries; degree III meniscus injury; chronic disease, such as diabetes mellitus; or peripheral neuropathy or vestibular dysfunction that could compromise proprioception. All patients were informed of the benefits and risks of ACLR with the LARS (LARS; Surgical Implants and Devices, Arc-sur-Tille, France), or hamstring tendon grafts. Eligible patients assured their compliance for participation in this study and provide written informed consent.

Block randomization was accomplished with a random number generator software program (SPSS version 22; IBM Corp) to ensure equal numbers in each group. The patients were sequentially allocated into one of two surgical groups based on the results of the generated random number table. It was not technically feasible to blind the surgeon to the participants’ allocated group.

Surgical procedures and patient rehabilitation

All procedures were assisted and performed by the same surgeons team of arthroscopy, who perform more than 100 LARS ligament surgeries every year. Routine arthroscopic inspection was performed through lateral and medial infrapatellar portals using a 30° oblique arthroscope, with the knee bent at 90°. Routine arthroscopic debridement was performed on all patients, and the details are as follows.

All autograft ACLRs used the anatomic single-bundle central ACLR technique. The ACLRs were ipsilateral six-stranded semitendinosus and gracilis tendon autografts. The ACL remnants were always preserved. The tibial tunnel was drilled at a point 2 mm anterior and 2 mm medial to the center of its attachment from the medial aspect of the proximal tibia, and the femoral tunnel was centrally located between the femoral attachments of the anteromedial and posterolateral bundles of the ACL. Tunnel size and dilation were based on the composite graft size. Femoral fixation was achieved with a Rigidloop (MiTek, US), and tibial fixation was accomplished with an INTRAFIX interference screw (MiTek, US).

All ACLRs with LARS were undertaken following isometric surgical principles, which have been previously described ^[20]. The ACL remnants were also always preserved, and the LARS ligament was inserted through it toward the resident’s ACL ridge. The intra-articular point of the tibial tunnel was positioned at the anteromedial part of the tibial footprint of the ACL from the anteromedial tibial cortex using a tibial aimer. The tibial tunnel was drilled with a cannulated reamer matching the diameter of the graft (7.5 mm). The femoral tunnel was positioned at the 11 o’clock position on the right knee and at nearly the 1 o’clock position on the left knee. The LARS ligament was inserted into the intra-articular joint through the ACL stump, and the longitudinal free fibers were retained in the joint. This ensured that the full range of knee motion was achieved and that there was no impingement among the LARS artificial ligaments, the notch, and the posterior cruciate ligament. Afterwards, the LARS graft was fixed by two titanium interference-fit screws (LARS; Surgical Implants and Devices, Arc-sur-Tille, France).

All patients were rehabilitated using the same postoperative rehabilitation protocol for the operated knee and were assessed in a similar manner. There was no deviation from the study protocol in any patient. Quadriceps isometric closed kinetic-chain exercises and straight leg raises were initiated as early as possible after ACLR. Knee flexion began from 45° within 2 weeks after the operation and increased gradually to complete flexion and extension within 4 weeks. Thereafter, the patients stopped using crutches. All patients were trained on balance rehabilitation exercises after their first proprioception evaluation (12 weeks after ACLR). The exercises included a single leg stance with the patient’s eyes open then closed for 5 minutes as a set and step-up exercises (anterior, lateral, and posterior) for both the uninvolved and involved leg ^[21].

Evaluation

Data on patient age, gender, affected side, body mass index (BMI), cause of injury, and time from injury to ACLR surgery were collected. Physical examinations, including the Lachman test, pivot-shift test, and KT-1000 arthrometer measurement (MEDmetric Corporation, San Diego, CA), were performed to evaluate knee laxity. Subjective clinical assessment was obtained using the International Knee Documentation Committee (IKDC) subjective knee evaluation form and the Lysholm Knee Scale.

Evaluation of proprioception was performed on both knees of all patients 3 and 12 months after ACLR using the JPS method (CMV AG, Dübendorf, Switzerland) according to the modified Barrack method ^[16]. In preparation for the measurement, the patients were seated in a neutral angle of lumbar flexion with the popliteal fossa situated approximately 5 cm from the edge of the seat. The JPS was measured at two angles (45° and 75°) from full extension (0°) to flexion ^{[16, 22]}. The tested leg was bent by a motor-driven rotational transducer interfaced with a computer to measure the reaction time of the passive motion. When the knee flexed to a predefined angle (45° or 75°), the leg was held in position for a few seconds, and the patient was asked to remember that position. The knee was returned to the starting position and then moved again by the motor at the same speed. When the patient felt that the leg was in the same position as before, the patient stopped the movement of the machine with a switch. The difference in the angle between the first perceived position and the second position (angular error) was measured. The JPS of both knees was measured three times for every patient (Fig. 2).

Statistical analysis

Data are shown as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS (version 22; IBM Corp). Levene’s test was used to evaluate whether there was equal variance in the continuous variables between the two groups. The chi-squared test was used to assess categorical variables, and Student’s t-test was used to assess continuous variables with normal distributions. A two-sided P < .05 was considered statistically significant.

Although 61 patients met the inclusion criteria for participation in the present study, 17 of these patients did not agree to the randomization of graft selection and were excluded, 4 of these patients were lost to follow up and were excluded. Thus, 40 patients with ACL rupture were recruited for this study, including 9 women and 31 men with a mean age of 35.5 years (range, 17–51 years). The autograft group using a six-strand ipsilateral hamstring autograft (semitendinosus and gracilis tendons) included 20 patients, 14 men and 6 women. The LARS group included 20 patients, 17 men and 3 women.

Before ACLR, there were no significant differences between the groups for the number of patients, gender distribution, age, BMI, Lysholm Scale score, or IKDC score (Table 1). However, the time between the injury and ACLR for the autograft group was significantly longer than that for the LARS group.

Table 1

Demographic and Clinical Characteristics of Participants
Characteristic	Autograft group (n = 20)	LARS group (n = 20)	P value*
Age (years)	37 ± 12(20–51)	36 ± 10(17–48)	0.41
Male /Female(n)	14/6	17/3	0.16
BMI (kg/m²)	24.3 ± 4.7	24.9 ± 5.7	0.81
Lysholm Scale score	52.5 ± 10.5	54.5 ± 8.7	0.55
IKDC score	47.8 ± 5.7	51.2 ± 8.6	0.74
Time from injury to ACLR operation (months)	18.4 ± 8.5	10.8 ± 6.1	< 0.05
Data were expressed as mean ± SD (range) unless otherwise indicated; ACLR, anterior cruciate ligament reconstruction; BMI, body mass index; IKDC, International Knee Documentation Committee; SD, standard deviation.
*Calculated by Student’s t-test for continuous variables and x² test for categorical variables between two groups.

A greater error in proprioception was detected using the JPS test for ACLR knees than for intact knees. Given that each patient may have a different proprioception baseline, each patient’s contralateral knee was used as the baseline to calculate the JPS deficit (JPSD). There was no significant difference in the loss of proprioception between the LARS group and the autograft group at either 3 or 12 months after the ACLR operation in the 45° or 75° flexion JPS test. However, for both the 45° and the 75° flexion JPS tests in the autograft group, the JPSD was significantly greater 3 months after ACLR than at 12 months after ACLR. Similar results were observed for the LARS group (Table 2 and Table 3).

Table 2

45° Flexion Joint Proprioception Sense Deficit in the Autograft and LARS Groups 3 and 12 Months After ACLR
Time of evaluation (months)	45°flexion		P value*
Time of evaluation (months)	3	12	P value*
Autograft group, degrees	2.81 ± 1.41	1.87 ± 0.96	< 0.05
LARS group, degrees	2.16 ± 0.85	1.65 ± 0.73	< 0.05
P value*	0.19	0.21
Data were expressed as mean ± SD (range) unless otherwise indicated; ACLR, anterior cruciate ligament reconstruction; LARS, Ligament Advanced Reinforcement System; SD, standard deviation.
*Calculated by Student’s t-test between two groups.

Table 3

75° Flexion Joint Proprioception Sense Deficit in the Autograft and LARS Groups 3 and 12 Months After ACLR
Time of evaluation (months)	75°flexion		P value*
Time of evaluation (months)	3	12	P value*
Autograft group, degrees	2.74 ± 1.13	1.77 ± 0.79	< 0.05
LARS group, degrees	2.42 ± 0.79	1.73 ± 0.56	< 0.05
P value*	0.45	0.59
Data were expressed as mean ± SD (range) unless otherwise indicated; ACLR, anterior cruciate ligament reconstruction; LARS, Ligament Advanced Reinforcement System; SD, standard deviation.
*Calculated by Student’s t-test between two groups.

The JPSD in patients whose time from injury to operation was more than 1 year was greater than that for patients whose time to ACLR was less than 1 year. Therefore, we reanalyzed the data by grouping the patients according to the time from injury to operation as more than 1 year (18 patients) and less than 1 year (22 patients). There was no statistically significant difference between these two groups in the number of patients, gender distribution, age, or BMI. Although the Lysholm Scale and IKDC scores of the group receiving ACLR more than 1 year after injury appeared lower than those in the group receiving ACLR less than 1 year after injury, neither score was significantly different (Table 4).

Table 4

Demographic and Clinical Patient Characteristics Analyzed by Time From Injury to ACLR Greater or Less Than 1 Year
Characteristic	Time from injury to ACLR operation(years)		P value*
Characteristic	> 1(n = 18)	< 1(n = 22)	P value*
Age (years)	35 ± 10(17–49)	37 ± 12(20–51)	0.52
Male /Female(n)	13/5	18/4	0.16
BMI (kg/m²)	25.1 ± 4.3	24.8 ± 4.9	0.78
Lysholm Scale score	47.2 ± 8.3	54.1 ± 9.6	0.47
IKDC score	49.8 ± 16.3	52.6 ± 18.7	0.74
Data were expressed as mean ± SD (range) unless otherwise indicated; ACLR, anterior cruciate ligament reconstruction; BMI, body mass index; IKDC, International Knee Documentation Committee; SD, standard deviation.
*Calculated by Student’s t-test for continuous variables and x² test for categorical variables between two groups.

A statistically significant difference was found between the groups with more and less than 1 year from injury to ACLR at both the 45° and 75° flexion JPS tests 3 months after ACLR; however, there was no significant difference between these groups for these measures 12 months after ACLR. The JPSDs from both the 45° and 75° flexion JPS tests for both groups 3 months after ACLR were significantly higher than those 12 months after ACLR (Table 5 and Table 6).

Table 5

45° Flexion JPS Deficits in Patients Analyzed by More or Less Than 1 Year From Injury to ACLR, Assessed 3 and 12 Months After ACLR
Time of JPS evaluation (months)	45°flexion		P value*
Time of JPS evaluation (months)	3	12	P value*
ACLR more than 1 year after injury	3.15 ± 1.05	1.96 ± 1.13	< 0.05
ACLR less than 1 year after injury	1.76 ± 0.72	1.45 ± 0.87	< 0.05
P value*	< 0.05	0.46
Data were expressed as mean ± SD (range) unless otherwise indicated; ACLR, anterior cruciate ligament reconstruction; JPS, joint position sense.
*Calculated by Student’s t-test between two groups.

Table 6

75° Flexion JPS Deficits in Patients Analyzed by More or Less Than 1 Year From Injury to ACLR, Assessed 3 and 12 Months After ACLR
Time of JPS evaluation (months)	75°flexion		P value*
Time of JPS evaluation (months)	3	12	P value*
ACLR more than 1 year after injury	2.72 ± 1.04	1.85 ± 0.02	< 0.05
ACLR less than 1 year after injury	1.80 ± 0.87	1.37 ± 0.54	< 0.05
P value*	< 0.05	1.10
Data were expressed as mean ± SD (range) unless otherwise indicated; ACLR, anterior cruciate ligament reconstruction; JPS, joint position sense.
*Calculated by Student’s t-test between two groups.

At the 1-year follow-up examinations, the results measured using the KT-1000 arthrometer showed that over 90% of the patients in both groups had side-to-side differences of 2 mm or less. In addition, the lateral pivot shift test showed no significant difference between the two ACLR groups (all were negative). However, this measure had improved compared with that obtained preoperatively. In the final test at the 1-year follow-up, the mean Lysholm Scale scores between the autograft (91.3 ± 3.8) and LARS (90.6 ± 5.2) ACLR groups were not significantly different; however, compared with their preoperative Lysholm Scale scores, all patient Lysholm Scale scores had significantly improved (P < 0.05). Moreover, all patients achieved full range of motion, and there were no complications, including no infection or early graft failure, within 12 months of ACLR.

Proprioception has been defined as the afferent information arising from the internal peripheral area of the body and contributing to postural control, joint stability, and specific conscious sensations^[23]. The proprioceptive deficit of a knee with a ruptured or nonfunctional ACL is well documented ^[24–28]. Assessments of TDPM and JPSD have been widely used to evaluate the changes in proprioception after ACL injury. Significant differences in both TDPM and JPS between ACL deficient and control knees have been reported. A recent meta-analysis by Kim et al. included 16 studies to compare proprioception in ACL-injured knees with the contralateral intact knees and found that proprioception as assessed by the sense of joint movement (kinesthesia) and joint position was decreased in the ACL-injured knees ^[29].

Much more attention has been paid to proprioception related to rehabilitation and gait than to that associated with the injury. To improve long-term function, physicians have established rehabilitation programs for patients that focus not only on range of motion and strengthening exercises but also on proprioceptive and neuromuscular control drills ^[30–33]. Sebastian Müller et al. showed that a functional neuromuscular training program was more effective than traditional muscle strength rehabilitation programs in improving function ^[33]. The results from all these aforementioned studies indicated that appropriate proprioception exercises would be expected to achieve significant improvements in knee proprioception and function in patients following ACLR.

The present study found no significant difference in the loss of proprioception between the LARS and the autograft groups 3 months after ACLR. The proprioception of patients in both groups was significantly improved after rehabilitation exercises, including performing a single leg stance with open then closed eyes for 5 minutes as a set and step-up exercises (anterior, lateral, and posterior) for the uninvolved and involved leg for 9 months. The present study also found no significant difference in proprioception between the two graft groups 12 months after ACLR. These results indicate that ACLR with either an autograft or a LARS artificial graft is safe and effective and that proprioception in patients with a LARS artificial ligament could be recovered to same level as that in patients with an autograft after appropriate exercises.

Previous studies have suggested that the knee proprioception deficit was associated with the disruption of mechanoreceptors within the ligament and subsequent loss of proprioceptive feedback ^[34–36]. However, Sha et al. used immunohistochemical assays to show that there was no significant quantitative variation in the residual mechanoreceptors throughout the injury duration ^[37]. Another study showed that the mechanoreceptors existed not only in the ligaments but also in the articular capsule, the collateral posterior cruciate ligaments, and muscles ^[38]. Trieb et al. found that instead of normal ligament tissue 6 months after ACLR with LARS, there were only fibroblasts and some endothelial cells surrounding the ligament fibers ^[39]. Therefore, after ACL rupture, the knee proprioception deficit may have a closer relationship with the abnormal neurologic output from the articular capsule, muscle, and other soft tissue than with the ACL itself. The results of our study indicated that proprioception recovery in the LARS group was similar to that in the autograft group. Therefore, the knee proprioception recovery after ACL rupture may be related primarily to the compensation of articular capsule, muscle, and other soft tissue.

The analysis of the data based on the time from injury to ACLR showed that patients with more than 1 year from injury to operation had a greater proprioception deficit 3 months after the ACLR than those with less than 1 year from injury to operation. This result indicates that early treatment after injury may improve postoperative knee function sooner. Nevertheless, 12 months after ACLR, there was no significant difference in proprioception between these two groups, indicating that knee proprioception in patients could substantially recover following appropriate rehabilitation exercises 12 months after ACLR.

Certain limitations of the present study should be addressed. First, only 40 participants were recruited, and there was a significant difference between the number of women and men participating (9 women and 31 men). Second, owing to technical limitations, we were unable to use a double-blind method. Third, the follow-up time was limited to 1 year; thus, additional research will be needed to observe the long-term effects of ACLR with different grafts on proprioception.

We found no significant difference in proprioception recovery between patients receiving a LARS graft and those receiving a hamstring tendon autograft 3 or 12 months after ACLR, and no complications, including infection or early graft failure, were associated with either ACLR method. These results suggest that ACLR with either an autograft or a LARS artificial graft is similarly safe and effective for knee proprioception recovery, which may be primarily associated with the compensation of the articular capsule, muscle, and other soft tissue.

ACL

Anterior cruciate ligament

ACLR

Anterior cruciate ligament reconstruction

LARS

Ligament Augmentation and Reconstruction System

JPS

Joint position sense

JPSD

Joint position sense deficit

TDPM

Threshold to detection of passive motion

BMI

Body mass index

IKDC

International Knee Documentation Committee

Standard deviation

Ethics approval and consent to participate

All procedures performed in this study involving human participants were in accordance with the ethical standards of the Shanghai University of Sport Research Ethics Committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the ethics committee of Shanghai University of Sport Research. Informed consent was obtained from each patient or their legal guardians. All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication

Not applicable, as no identifying personal information is included in this manuscript.

Availability of data and materials

The data contributing to this article may be made available upon request by sending an e-mail to Changli Xu or Tianze Liu.

Competing interests

All authors declare that they have no competing interests.

Funding

This work is supported by the National Natural Science Foundation of China (Grant nos. 82171383) and the “Discipline Construction Climbing 234 Plan” of Changhai Hospital of Naval Medical University (Second Military Medical University) (Grant nos. 2019YXK055).

Authors' contributions

Changli Xu and Zhiwei Wang contributed to the study conception and design. Changli Xu, Tianze Liu and Miao Wang carried out the data collection and drafted the manuscript. Tianze Liu, Chang Liu, Bo Li and Suchi Qiao analyzed and interpreted the data. Qiujian Lian, Tongjiang Chen and Fengmei Chen substantively revised the manuscript. All authors read and approved the final version of the manuscript.

Acknowledgements

We appreciate the contribution of all patients and their guardians and thank all staff from the participating departments and clinics.

Authors' information

Changli Xu, Tianze Liu and Miao Wang contributed equally to this work，these authors are co-first authors.

Affiliations

1. Department of Orthopedics, Changhai Hospital, Naval Medical University, Shanghai 200433, People’s Republic of China.

Changli Xu, Tianze Liu, Miao Wang, Chang Liu, Bo Li, Qiujian Lian, Tongjiang Chen

2. Department of Orthopedics, The Third Affiliated to the Naval Military Medical University, Shanghai 201805, People’s Republic of China.

Changli Xu, Miao Wang, Fengmei Chen, Suchi Qiao, Zhiwei Wang

3. The fifth outpatients department, 980 Hospital of joint service support force，Shijiazhuang 050083, People’s Republic of China.

Changli Xu

Correspondence authors

Correspondence to Fengmei Chen, Suchi Qiao or Zhiwei Wang.

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No competing interests reported.

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Comparison of Proprioception Recovery Following Anterior Cruciate Ligament Reconstruction Using an Artificial Graft Versus an Autograft

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Abstract

Background

Material and Methods

Results

Conclusion

Figures

Background

Material And Methods

Patient selection and randomization

Surgical procedures and patient rehabilitation

Evaluation

Statistical analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Competing Interests

Status:

Version 1