Effects of a low, medium, and high-intensity aquatic physiotherapy protocol on functional and biochemical parameters in individuals with knee osteoarthritis: protocol for a crossover randomized controlled trial

Trial design

This is an interventional transversal study, randomized clinical trial, crossover type with three arms: low, medium, and high-intensity aquatic physiotherapy.

This clinical intervention protocol follows the recommendation from the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT). ^{32 , 60} The study sample, defined according to criteria, will be submitted to a single session of each one of the following modalities of aquatic physiotherapy: (a) low-intensity, (b) medium-intensity, and (c) high-intensity. Among each modality, a minimum of a 15-day wash-out period will be respected, in accordance with Figure 1.

Figure 1. Flow of interventions and assessment parameters.

For this study, 15 participants will be recruited. The sample size calculation revealed a power of >0.80 and adopting an alpha value of 0.05 in order to detect the differences among groups and the differences among the repeated measures (within the subject effect). The effect size calculation was based on means and standard deviations, and for our main dependent variables, these were reported by previous investigations: Germanou ³³ through the antioxidant levels and Nascimento ³⁴ through the MMP-2 and MMP-9 analysis between the control and intervention group, in this case, it is represented by patients with knee osteoarthritis.

The assessment parameters used in each variable will consist of instruments that allow for the assessment of the variations in the MMP-3 concentration, oxidative stress, pain, edema, and flexibility. In addition, a self-perception questionnaire, developed by the authors, will be applied to assess self-perception. It consists of a Likert scale where the patient must classify their perception of well-being between zero and 10, where zero represents the worst feeling of well-being and 10 represents the best feeling of well-being. The data collection form, informed consent form and patient identification form can be found as Extended data. ⁶⁰ The trial has been registered on ClinicalTrials.gov (NCT05610696, 18/01/2023).

Study setting

The study population corresponds to people with knee osteoarthritis. The population will be recruited from physiotherapy services from University Feevale.

The protocol will be performed in the facilities of the Aquatic Physiotherapy sector. The swimming pool has accessibility via stairs and a ramp with appropriate handrail support. Heating is provided by an electronic boiler fed with liquefied petroleum gas that guarantees minimum temperature variations (33°C and 34°C) measured with a submerged mercury column thermometer. The swimming pool depth varies between 100 and 150 cm, transitioned by means of a ramp; the entire swimming pool is lined with non-slip tiles. The swimming pool also has two hydromassage spaces on one of the borders. Each one is equipped with hydromassage jets, one in the trunk support region and two on the sides of this area.

Eligibility criteria

The following eligibility criteria will be considered: being over 50 years old and diagnosed with osteoarthritis in at least one knee according to the American College of Rheumatology ³⁵ criteria, referring to pain and at least one dysfunction for at least six months, absence of physical limitation that prevents the performance of the exercises protocol, have Kellgren and Lawrence classification between I and IV ³⁶ and independent gait. Subjects who present autoimmune inflammatory conditions, use of psychotropic, immunosuppressant, or steroids drugs, consumption of antioxidants (vitamin E, statins, or uric acid reducers), alcohol and tobacco, progressive ankylosis states, the coexistence of neurological lesions, infections or skin diseases that contraindicate swimming pool immersion, surgical procedures on the knee joint in the last 12 months (excluding meniscectomy or arthroscopy), lower limb articular prosthesis, presence of inflammatory or infectious disease, uncontrolled systemic arterial hypertension, uncontrolled diabetes, chronic obstructive pulmonary disease and history of ischemic heart disease, self-reported aquatic phobia, and chlorine allergy will be excluded from the study. For control purposes, a reference group will be constituted with age matching and exclusion criteria identical to the experimental group.

Outcomes

Initially, all participants will be interviewed and evaluated in relation to the degree of osteoarthritis and functionality. Information regarding the patient data will be confirmed via interview, considering data such as complete name, date of birth, age, affected knee(s), duration of pain, arterial blood pressure, list of medications in use, consumption of tobacco or alcohol, associated diseases, if they are undergoing physiotherapy treatment or regular physical activity.

The classification regarding the degree of osteoarthritis will be performed by analyzing the radiograph of the affected knee according to the method of Kellgren and Lawrence. The radiographs will be analyzed, the degree of osteoarthritis will be identified with the negatoscope support, and the images in the digital form will be displayed on a computer screen, considering the classification expressed in degrees ranging from zero to IV.

Subsequently, the identification of functionality will be assessed according to the Brazilian version of the Knee Injury and Osteoarthritis Outcome Score (KOOS). The KOOS questionnaire was developed in the 1990s as an instrument to evaluate the patient’s perception of their knee and associated problems. Since its first publication in 1998, the psychometric properties of KOOS were evaluated in a systemic review of 37 articles demonstrating adequate validity in its use for the study population. ³⁷

Regarding the pre- and post-intervention assessment instruments, all participants will have the following parameters evaluated: concentration of MMP-3 and GPx, pain, edema, flexibility, and self-perception of the general well-being. In addition, it should be noted that in the 24-hour-follow-up, only the biochemical concentrations and the self-perception questionnaire will be reevaluated.

The biochemical variables will be measured in peripheral blood from the concentrations of MMP-3 and GPx. The levels of MMPs will be measured based on the Enzyme-Linked Immunosorbent Assay (ELISA) methodology, which is based on the antigen-antibody interaction and will be performed according to the kit manufacturer’s instructions. MMP-3, a constituent of the stromelysin family that is an important mediator responsible for the degradation of collagen type II, IX, and XI, ^{13 , 14} will be considered for this analysis. Oxidative stress will be measured by analyzing the peripheral blood from percutaneous punction. The sample collection time will be performed according to the study by Nascimento ³⁴ and will be in accordance with the following times: baseline moment (immediately before the intervention), moment 1 (immediately after the intervention), and moment 2 (24 hours after the intervention).

The assessment of the degree of pain will be performed using the visual analogue pain scale, ³⁸ from a scale graduated from zero to 10, in which zero corresponds to cases without pain and 10 corresponds to the worst imaginable pain.

The assessment of edema will be performed based on the measurement of the circumference of the affected knee(s). The measure will be performed with the patient relaxed in dorsal decubitus and the circumference will be determined with the knee in extension, based on the proximal distance of 1 cm from the base of the patella. ³⁹ For that, the same measuring tape will be used for all measurements.

The flexibility will be assessed by measuring the amplitude of the active and passive movement for the knee flexion and, for that, a conventional goniometer will be used. The goniometer will be used because it has a low cost, is easy to handle, allows the assessments to be performed quickly, and the measurements and movement amplitude for the knee joint are valid and reliable. ⁴⁰ For the assessment, the knee will be free of clothing, and the patient will be positioned in dorsal decubitus with the hip articulation at 90° of flexion. The fixed arm of the goniometer will be positioned parallel to the thigh with an orientation towards the greater trochanter of the femur and the mobile arm will be parallel to the lateral face of the fibula, directed towards the lateral malleolus. The movement reading will be performed while maintaining the final possible position of the arc of movement where the highest reading degree represents the assessed measurement. ⁴¹ All procedures will be performed by a single physiotherapist.

Interventions

The subjects of this study will be submitted to different interventions of aquatic physiotherapy systematized as follows: program A (low-intensity protocol), program B (medium-intensity protocol), and program C (high-intensity protocol).

The access to the immersion will be from a ladder, and a period of 5 minutes will be allowed for acclimatization to the liquid environment. Considering that the intervention proposals aim to provide different levels of articular overload, the speed and time of execution will be gradually variable.

Program A will use the principles of Ai Chi^® as this model is defined as a strategy of low intensity and consists of a sequence of slow movements. ⁴² For that, a sequence of adapted exercises ⁴³ will be performed, according to Table 1.

Table 1. Program A: low-intensity protocol.

The individuals in the images are students of the Physiotherapy course who consented and authorized their image.

Exercise	Periodization	Description
	4 minutes	Antero-posterior and latero-lateral walk.
	4 minutes, 2×2 minutes	Horizontal adduction and abduction of the shoulders alternately with rotation of the trunk to the left and right, aiming at transferring the weight in each of the lower limbs.
	4 minutes, 2×2 minutes	In the sitting position, perform flexion and extension of both knees alternately.
	4 minutes, 2×2 minutes for each articulation	In orthostatism, perform hip circumduction.
	4 minutes, 2×2 minutes for each articulation	In orthostatism, perform abduction of both hips alternately.
	4 minutes, 2×2 minutes for each articulation	In orthostatism, perform flexion of both hips alternately.
	4 minutes, 2×2 minutes for each articulation	In orthostatism, initial position in hip flexion at 90°, perform knee flexion and extension.
	4 minutes, 2×2 minutes for each articulation	Elevation of the lower limb forward (single leg support) during the horizontal shoulder abduction and adduction phase. Return to bipedal support during the horizontal shoulder abduction and adduction phase.
	4 minutes, 2×2 minutes for each articulation	Hip flexion (single leg support) concomitant with shoulder extension followed by hip extension (bipedal support) concomitant with shoulder flexion.
	4 minutes, 2×2 minutes for each articulation	Cross the forearms in front of the umbilical scar, followed by the horizontal flexion together with the movement of crossing one lower limb over the other.
	5 minutes	Immersion in the whirlpool bath
Fifteen seconds rest between each exercise Total program time: approximately 45 minutes

As a starting position to perform the exercises, the participants must remain in the semi-flexion position of their knees so that the water line must remain at shoulder height throughout the procedure. All the exercises will be performed according to the established time, and a 15-second rest time will be added between each exercise or series. As a manner of manipulating the intensity of the exercise at low intensity, the movement speed will be standardized according to the subjective sensation of effort according to Borg 6-20, ⁴⁴ with zones of interest from 6 (extremely light) to 9 (very light). In this intervention modality, we will seek to understand the impact that the physiological effects of immersion in heated water pose on the studied variables.

Program B will consist of a sequence of exercises adapted from previous works ^{45 , 46} composed of warm-up, fortification, and cool-down activities. Aquatic steppes platforms will be used to ensure that all participants, when performing the exercises in orthostatism, will maintain the waterline level approximately at the level of the xiphoid appendix. For this program, the exercises will be performed at a speed that guarantees a comfortable water level for the activity, being the effort zones of interest from 10 (fairly light) to 14 (somewhat hard).

The warm-up sequence consists of four different active moves performed at maximum movement amplitude and contributes to neuromuscular activation.

After the warm-up, the participants will be submitted to the fortification program, which consists of a sequence of seven exercises aiming to promote muscular activation. The participants will be encouraged to perform the movement at a comfortable speed with the maximum possible movement amplitude. During sitting exercises, the need to maintain the lumbar spine in a neutral position will be emphasized in order to avoid overloading this segment.

To finish program B, as a way to calm down, a sequence of three activities will be performed, including a period in the hydromassage area, according to Table 2.

Table 2. Program B: medium-intensity protocol.

The individuals in the images are students of the Physiotherapy course who consented and authorized their image.

Warm-up
Exercise	Periodization	Description
	4 minutes	Antero-posterior and latero-lateral walk.
	4 minutes	Antero-posterior and latero-lateral walk with obstacles over 30-cm-steppes.
	1 minute	Sitting and standing up.
	1 minute	Skipping - running on the same place.
Total warm-up time: approximately 10 minutes
Fortification
	3 minutes	In orthostatism with unipedal support of the lower limb contralateral to the moved segment, perform hip flexion and extension. The knee must remain in complete extension and the ankle in dorsiflexion. The lumbar spine must remain in a neutral position.
	3 minutes	In orthostatism, perform hip abduction and adduction. The knee must remain in complete extension with the ankle in dorsiflexion. The movement must contemplate the maximum amplitude with the maintenance of the pelvis and lumbar spine in a static position.
	3 minutes	In orthostatism, perform the movement of knee flexion and extension. The movement must contemplate the maximum flexion and extension with the maintenance of the lumbar spine in a neutral position.
	3 minutes	In orthostatism, perform the wide movement of hip extension and knee flexion followed by hip flexion and knee extension (kick movement).
	3 minutes	With the help of floats, perform the bicycle movement (alternating knee and hip flexion) aiming at the anterior displacement.
	3 minutes	Patient in a supine position with the help of floats, perform the backstroke leg kick aiming at the cranial displacement.
	3 minutes	In orthostasis, perform the slow walking movement, simulating a Nordic walk, sliding the feet on the bottom of the pool, however, without moving.
Program B fortification program time: approximately 21 minutes.
Cool down
	1 minute	Antero-posterior and latero-lateral walk.
	30 seconds on each side	Active stretching of the gluteus maximus.
	30 seconds on each side	Active stretching of the quadriceps.
	30 seconds on each side	Active stretching of the hamstring.
	30 seconds on each side	Active stretching of the gastrocnemius.
	2 minutes	Immersion in the whirlpool.
Total cool down time: approximately 8 minutes.

In program C (high-intensity protocol), the same sequence of exercises stipulated for program B will be respected, however, the quantity of work will be modified based on the variables time and speed of the performance of the movement. ⁴⁷ Fortification activities will be performed for 6 minutes, and the speed must be adjusted in order to stimulate the performance of the movement for the subjective sensation compatible with the zone of interest 15 (hard) to 18 (very hard). In the same way, walking activity will be replaced by running activity which amounts to an approximate time of 39 minutes.

The reference group will be submitted to the same aquatic physiotherapy protocols, however, only the biochemical parameters and the self-perception questionnaires will be evaluated.

Recruitment

The population will be recruited from the physiotherapy services of Universities. The study will also be disseminated in print and digital media, social networks of the researcher, the university, the academic directory of health courses, the Medical Leagues and institutional pages.

Assignment of interventions

Allocation: Participants will be allocated randomly to establish the sequence within each group, following a computer-generated randomization schedule from randomize.com, as exemplified by the flowchart in Figure 1.

Blinding: This study is classified as single-blinded, indicating that the researchers responsible for conducting the assessment instruments and laboratory analysis procedures will be distinct from those administering the intervention protocols.

Data collection, management, and analysis

Data collection methods: The interviews, evaluation of radiological images, application of instruments and measurements of pain, edema, and flexibility will be carried out only by a single qualified, trained physiotherapist, with familiarity with the instruments and with adequate training. Blood sample collection will be performed by a nursing professional familiar with this procedure. Laboratory analyzes will be performed by another researcher qualified biomedical pharmacist. The application of the intervention protocols will be carried out by two physiotherapists trained in aquatic physiotherapy and who will not participate in the interviews, radiological analysis and evaluation of edema, pain and flexibility.

Data management: All data will be entered electronically via Microsoft Excel. The study’s digital forms will be inserted and received in a cloud file with access only to the researcher. Participant files will be stored in numerical order and stored in storage for a period of 3 years after completion of the study. Modifications to data recorded in the database will be documented through the data breach system associated with your user identification code and password. Access to study data will be restricted. All reports will be prepared in such a way that no subject can be identified.

Statistical analysis

The resulting data will be tabulated, considering the absolute values and their respective standards. Information regarding the main outcome corresponding to the variations in the concentrations of MMP-3 and GPx in peripheral blood due to three different aquatic physiotherapy interventions will be comparatively evaluated among the distinct interventions and among the different times (in pre- and post-intervention moments).

The information on the outcome regarding pain, edema, and flexibility will be comparatively assessed among the different types of intervention. Statistical analyses will be performed in the SPSS software and will be considered as a significance level when p>0.05 through the repeated measures ANOVA statistical test.

Monitoring

Data monitoring

We perceive the establishment of a Data Monitoring Committee as unnecessary due to the brief duration of this study and the established safety levels associated with aquatic physiotherapy for patients diagnosed with knee osteoarthritis.

Harms

In our study, an adverse event will be defined as any unfavorable functional occurrence in an individual consistently linked to exercise intensity. Adverse events will be documented after the subject provides consent and enrollment in the study. Researchers will evaluate the connection between an event and the exercise intensity of the study protocol, based on a temporal relationship, in addition to assessing whether the event is unexpected or unexplained. The study will meticulously track the subsequent protocol-associated adverse effects: heightened pain, inflammation, and diminished flexibility immediately post-intervention and at the 24-hour mark through patient examination. For any circumstance that may necessitate urgent medical support, the emergency medical service of the Aquatic Physiotherapy department will be activated to ensure the requisite and appropriate assistance.

Auditing

Regarding this matter, we find it unnecessary due to the nature of our single-center study, which involves a limited sample size and employs a therapeutic approach that has been well-established as safe.

Ethics and dissemination

Ethics approval and consent

This research was approved by the Research Ethics Committee of Federal University of Health Sciences in Porto Alegre in 15/05/2020 founder the number 4.030.865 and Presentation Certified for Ethical Appreciation (CAAE) number 29447220.5.0000.5345. Patients will be invited to participate in the study voluntarily, and they must consent to their participation by formalizing the Free and Informed Written Consent according to established by resolution number 466 from the National Health Council. ⁴⁸ The study is planned to take place at University Feevale, and ethical approval has been obtained from the Research Ethics Committee of the Federal University of Health Sciences in Porto Alegre. This choice of institution for ethical approval is due to a strategic partnership between the two universities for this research endeavour. The partnership was established because our research advisor is affiliated with the Department of Physical Therapy at the Federal University of Health Sciences, making this institution the natural choice for ethical oversight. This partnership ensures that both institutions are jointly responsible for the successful execution of the study and ethical compliance is maintained throughout. The University Feevale is a co-participating institution in the research and, as such, declares its compliance with Resolution CNS 466/2012, which pertains to Brazilian regulations for research involving human subjects.

Protocol amendments

While the prescribed protocol has been deemed safe, having undergone pilot interventions within the aquatic physiotherapy department of the university, any modifications that might impact the study’s execution will necessitate renewed consideration by the Ethics Committee.

Confidentiality

Participant study data will not be disclosed beyond the study without explicit written consent from the participants. This confidentiality is upheld through the previously approved Informed Consent Form.

Access to data

Sole access to the datasets will be granted to the Principal Researcher. Project data shall be securely stored within password-protected files. To ensure privacy, data shared with project team members will be stripped of any participant-identifying information.

Ancillary and post-trial care

The responsible researcher will promptly provide comprehensive support to research participants concerning potential complications and resulting implications. Similarly, considering the research’s conduct within an accredited aquatic physiotherapy service, the service will consistently uphold any requisite therapeutic follow-ups after the study.

Dissemination

The study will be disseminated in printed and digital media, on the researcher, university, and the academic directory of health courses social media, in the Medicine Leagues, and on institutional pages.

Study status

Not yet recruiting.