The research question is if there is a dose-response relationship between blood biomarkers for articular cartilage and the magnitude of physiological load. To investigate this load induced biomarker change, the participants will complete a 30-minute walking test under three loading conditions: A: 80%, B: 100% C: 120% of their respective bodyweight (Fig. 1).

The main biomarker we are interested in is called cartilage oligomeric matrix protein (COMP). Serum COMP levels are increased immediately after 30-min running but there is no dose-response relationship shown yet.

The current state of research in the field and of our own research clearly shows that the in vivo mechanobiology of articular cartilage is poorly understood. Previous results of changes in cartilage biomarker concentrations after ambulatory exercise raise the following question:

Do load-induced changes in blood biomarkers for articular cartilage (COMP) depend on the physiological load magnitude? To generate initial data for answering this question, we will establish the experimental framework to systematically study the in vivo mechanobiology of human articular cartilage to address the following aims.

• To obtain pilot data for investigating the in vivo dose-response relationship of weight-bearing and blood levels of mechanosensitive biomarker of articular cartilage using controlled load- bearing as experimental paradigm.
• To provide pilot data for comparing ambulatory biomechanics between the controlled load- bearing conditions.

The experimental setting consists of a 30-minutes walking stress test under three loading conditions: A: 80 %, B: 100% and C: 120% bodyweight. Unloading will be achieved through an unloading system and additional loading with an adjustable weight vest. Before and after the walking stress test, venous blood samples will be taken at five time points. The biomarker concentration is measured with a commercial available enzyme-linked immunosorbent assay (ELISA).

Hypothesis Our working hypothesis is that there is a dose-response relationship between the temporarily increased or decreased load and the load-induced change in COMP level in the blood serum. We assume that serum COMP concentration increases with increasing accumulated load on the knee cartilage simulated through the three different loading conditions.

Design The design of the pilot study is a controlled multimodal data collection (biological, biomechanical) with block randomization. Specifically, we will collect blood samples and biomechanical (kinematic and pressure) data. We will determine the relationship between the serum blood concentration and the biomechanical data.

Because this is a cross-over study, all participants will perform the same three walking stress tests but in random order. Each participant of the study will be tested for 2.5 hours on 3 separate days.

Method In this experiment, we will test 24 healthy participants (12 male, 12 female) on 3 test days under three different loading conditions in a randomized block design. On each test day, participants will complete walking stress test for 30-minutes with A: 80%, B: 100% and C: 120% of their bodyweight. Through the modified bodyweight we generate higher or lower forces on the knee cartilage, which should affect the COMP concentration in the blood serum.

To measure the change in serum COMP concentration, blood samples will be taken at five time points before and after the walking stress test. The blood samples are taken by a study nurse. For the venipuncture, a thin, sterile, disposable vein catheter is placed in the antecubital vein. The catheter stays in the vein for the entire 2.5 hours of the experiment. Blood samples will be collected and allowed to clot for 30-minutes. Serum will be separated and frozen in aliquots to -20°C within 1 hour of collection and then transferred for storage at -80°C until assayed. Serum biomarker concentrations will be determined using commercial ELISAs.

Inclusion Criteria:

• Age between 18 and 30 years
• being physically active (> 2/week)
• body mass index (BMI) <30 kg/m2

Exclusion Criteria:

• previous knee injury
• neuromuscular condition affecting gait

Masking is not possible because the experimental condition is obvious. Participants and testers cannot influence outcome parameters.