[Tissue biomechanics: connective tissue characterization : Cluster tissue biomechanics]. / Gewebebiomechanik: Charakterisierung von Bindegewebe : Cluster Gewebebiomechanik.

The various connective tissues of the body have different functions, which result from their specific structure and composition. The identification of this structure-function relationship is of great importance for various disciplines such as medicine, biology or tissue engineering. Connective tissue consists mainly of an extracellular matrix (ECM) and a limited number of cells. It is extremely adaptable because the activity of the cells remodels the composition and structure of the ECM in order to adapt the mechanical properties (functions) to the new demands (e.g. an increased mechanical stimulus).

Osteoclast-specific Plastin 3 knockout in mice fail to develop osteoporosis despite dramatic increased osteoclast resorption activity.

PLS3 loss-of-function mutations in humans and mice cause X-linked primary osteoporosis. However, it remains largely unknown how PLS3 mutations cause osteoporosis and which function PLS3 plays in bone homeostasis. A recent study showed that ubiquitous Pls3 KO in mice results in osteoporosis. Mainly osteoclasts were impacted in their function However, it has not been proven if osteoclasts are the major cell type affected and responsible for osteoporosis development in ubiquitous Pls3 KO mice. Here, we generated osteoclast-specific Pls3 KO mice. Additionally, we developed a novel polyclonal PLS3 antibody that showed specific PLS3 loss in immunofluorescence staining of osteoclasts in contrast to previously available antibodies against PLS3, which failed to show PLS3 specificity in mouse cells. Moreover, we demonstrate that osteoclast-specific Pls3 KO causes dramatic increase in resorptive activity of osteoclasts in vitro. Despite these findings, osteoclast-specific Pls3 KO in vivo failed to cause any osteoporotic phenotype in mice as proven by micro-CT and three-point bending test. This demonstrates that the pathomechanism of PLS3-associated osteoporosis is highly complex and cannot be reproduced in a system singularly focused on one cell type. Thus, the loss of PLS3 in alternative bone cell types might contributes to the osteoporosis phenotype in ubiquitous Pls3 KO mice.

Immobilization by 21 days of bed rest results in type II collagen degradation in healthy individuals.

OBJECTIVE: To investigate the effects of 21 days of bed rest immobilization (with and without exercise and nutrition interventions) on type II collagen biomarker concentrations in healthy individuals. DESIGN: Twelve healthy male participants (age 34.2 ± 8.3 years; body mass index 22.4 ± 1.7 kg/m²) were exposed to 6 days ambulatory baseline data collection (BDC), 21 days head-down-tilt bed rest (HDT, CON) + interventions (HDT + resistive vibration exercise (2 times/week, 25 minutes): RVE; HDT + RVE + whey protein (0.6 g/kg body weight/day) and bicarbonate supplementation (90 mmol KHCO3/day: NeX), and 6 days of re-ambulation (R) in a cross-over designed study. The starting HDT condition was randomized (CON-RVE-NEX, RVE-NEX-CON, NEX-CON-RVE). Blood and urine samples were collected before, during, and after HDT. Serum concentrations (s) of CPII, C2C, C1,2C, and urinary concentrations (u) of CTX-II and Coll2-1NO2 were measured. RESULTS: Twenty-one days of HDT resulted in increased sCPII (p < 0.001), sC2C (p < 0.001), and sC1,2C (p = 0.001) (highest increases: sCPII (+24.2% - HDT5), sC2C (+24.4% - HDT7), sC1,2C (+13.5% - HDT2). sC2C remained elevated at R+1 (p = 0.002) and R+6 (p < 0.001) compared to baseline. NeX led to lower sCPII (p < 0.001) and sC1,2C (p = 0.003) compared to CON. uCTX-II (second void and 24-hour urine) increased during HDT (p < 0.001, highest increase on HDT21: second void +82.8% (p < 0.001); 24-hour urine + 77.8% (p < 0.001). NeX resulted in lower uCTX-II concentrations in 24-hour urine (p = 0.012) compared to CON. CONCLUSIONS: Twenty-one days of bed rest immobilization results in type II collagen degradation that does not recover within 6 days of resuming ambulation. The combination of resistive vibration exercise and protein/bicarbonate supplementation minimally counteracted this effect.

Ablation of integrin-mediated cell-collagen communication alleviates fibrosis.

OBJECTIVES: Activation of fibroblasts is a hallmark of fibrotic processes. Besides cytokines and growth factors, fibroblasts are regulated by the extracellular matrix environment through receptors such as integrins, which transduce biochemical and mechanical signals enabling cells to mount appropriate responses according to biological demands. The aim of this work was to investigate the in vivo role of collagen-fibroblast interactions for regulating fibroblast functions and fibrosis. METHODS: Triple knockout (tKO) mice with a combined ablation of integrins α1ß1, α2ß1 and α11ß1 were created to address the significance of integrin-mediated cell-collagen communication. Properties of primary dermal fibroblasts lacking collagen-binding integrins were delineated in vitro. Response of the tKO mice skin to bleomycin induced fibrotic challenge was assessed. RESULTS: Triple integrin-deficient mice develop normally, are transiently smaller and reveal mild alterations in mechanoresilience of the skin. Fibroblasts from these mice in culture show defects in cytoskeletal architecture, traction stress generation, matrix production and organisation. Ablation of the three integrins leads to increased levels of discoidin domain receptor 2, an alternative receptor recognising collagens in vivo and in vitro. However, this overexpression fails to compensate adhesion and spreading defects on collagen substrates in vitro. Mice lacking collagen-binding integrins show a severely attenuated fibrotic response with impaired mechanotransduction, reduced collagen production and matrix organisation. CONCLUSIONS: The data provide evidence for a crucial role of collagen-binding integrins in fibroblast force generation and differentiation in vitro and for matrix deposition and tissue remodelling in vivo. Targeting fibroblast-collagen interactions might represent a promising therapeutic approach to regulate connective tissue deposition in fibrotic diseases.

Ablation of collagen XII disturbs joint extracellular matrix organization and causes patellar subluxation.

Collagen XII, belonging to the fibril-associated collagens, is a homotrimeric secreted extracellular matrix (ECM) protein encoded by the COL12A1 gene. Mutations in the human COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome leading to skeletal abnormalities and muscle weakness. Here, we studied the role of collagen XII in joint pathophysiology by analyzing collagen XII deficient mice and human patients. We found that collagen XII is widely expressed across multiple connective tissue of the developing joint. Lack of collagen XII in mice destabilizes tendons and the femoral trochlear groove to induce patellar subluxation in the patellofemoral joint. These changes are associated with an ECM damage response in tendon and secondary quadriceps muscle degeneration. Moreover, patellar subluxation was also identified as a clinical feature of human patients with collagen XII deficiency. The results provide an explanation for joint hyperlaxity in mice and human patients with collagen XII deficiency.

EMILIN1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis.

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.

Ablation of the miRNA cluster 24 in cartilage and osteoblasts impairs bone remodeling.

MicroRNAs (miRNAs) post-transcriptionally regulate cartilage and bone development and function, however, only few miRNAs have been described to play a role for cartilage to bone transition in vivo. Previously, we showed that cartilage-specific deletion of the Mirc24 cluster in newborn male mice leads to impaired growth plate cartilage development due to increased RAF/MEK/ERK signaling and affects the stability of the cartilage extracellular matrix on account of decreased SOX6 and SOX9 and increased MMP13 levels. Here, we studied how Mirc24 cluster inactivation in cartilage and osteoblasts leads to an increased bone density associated with defects in collagen remodeling in trabecular bone. No changes in osteoblast distribution were observed, whereas the number of osteoclasts was reduced and TRAP activity in osteoclasts decreased. Surprisingly, an increased level of cluster-encoded miR-322 or miR-503 raises Rankl gene expression and inactivation of the cluster in chondrocytes reduces Rankl expression. These results suggest that the Mirc24 cluster regulates Rankl expression in chondrocytes at the chondro-osseous border, where the cluster is mainly expressed to modulate osteoclast formation, bone remodeling and bone integrity.

Relationship between different serum cartilage biomarkers in the acute response to running and jumping in healthy male individuals.

The effect of physical activity on serum cartilage biomarkers is largely unknown. The purpose of the study was to systematically analyze the acute effect of two frequently used exercise interventions (running and jumping) on the correlation of seven serum biomarkers that reflect cartilage extracellular matrix metabolism. Fifteen healthy male volunteers (26 ± 4 years, 181 ± 4 cm, 77 ± 6 kg) participated in the repeated measurement study. In session 1, the participants accomplished 15 × 15 series of reactive jumps within 30 min. In session 2, they ran on a treadmill (2.2 m/s) for 30 min. Before and after both exercise protocols, four blood samples were drawn separated by 30 min intervals. Serum concentrations of seven biomarkers were determined: COMP, MMP-3, MMP-9, YKL-40, resistin, Coll2-1 and Coll2-1 NO2. All biomarkers demonstrated an acute response to mechanical loading. Both the COMP and MMP-3 responses were significantly (p = 0.040 and p = 0.007) different between running and jumping (COMP: jumping + 31%, running + 37%; MMP-3: jumping + 14%, running + 78%). Resistin increased only significantly (p < 0.001) after running, and Coll2-1 NO2 increased significantly (p = 0.001) only after jumping. Significant correlations between the biomarkers were detected. The relationships between individual serum biomarker concentrations may reflect the complex interactions between degrading enzymes and their substrates in ECM homeostasis.

Bisphenol A-Related Effects on Bone Morphology and Biomechanical Properties in an Animal Model.

Bisphenol A (BPA), which is contained in numerous plastic products, is known to act as an endocrine-disruptive, toxic, and carcinogenic chemical. This experimental series sought to determine the influence of BPA exposure on the femoral bone architecture and biomechanical properties of male and female Wistar rats. BPA was applied subcutaneously by using osmotic pumps. After 12 weeks, the bones were analyzed by micro-computed tomography (micro-CT) and a three-point bending test. Comparing the female low- and high-dose groups, a significantly greater marrow area (p = 0.047) was identified in the group exposed to a higher BPA concentration. In addition, the trabecular number tended to be higher in the female high-dose group when compared to the low-dose group (p > 0.05). The area moment of inertia also tended to be higher in the male high-dose group when compared to the male low-dose group (p > 0.05). Considering our results, BPA-related effects on the bone morphology in female Wistar rats are osteoanabolic after high-dose exposure, while, in male rats, a tendency toward negative effects on the bone morphology in terms of a reduced cross-sectional cortical area and total area could be demonstrated.

Sensitive asprosin detection in clinical samples reveals serum/saliva correlation and indicates cartilage as source for serum asprosin.

The C-terminal pro-fibrillin-1 propeptide asprosin is described as white adipose tissue derived hormone that stimulates rapid hepatic glucose release and activates hunger-promoting hypothalamic neurons. Numerous studies proposed correlations of asprosin levels with clinical parameters. However, the enormous variability of reported serum and plasma asprosin levels illustrates the need for sensitive and reliable detection methods in clinical samples. Here we report on newly developed biochemical methods for asprosin concentration and detection in several body fluids including serum, plasma, saliva, breast milk, and urine. Since we found that glycosylation impacts human asprosin detection we analyzed its glycosylation profile. Employing a new sandwich ELISA revealed that serum and saliva asprosin correlate strongly, depend on biological sex, and feeding status. To investigate the contribution of connective tissue-derived asprosin to serum levels we screened two cohorts with described cartilage turnover. Serum asprosin correlated with COMP, a marker for cartilage degradation upon running exercise and after total hip replacement surgery. This together with our finding that asprosin is produced by primary human chondrocytes and expressed in human cartilage suggests a contribution of cartilage to serum asprosin. Furthermore, we determined asprosin levels in breast milk, and urine, for the first time, and propose saliva asprosin as an accessible clinical marker for future studies.

Hemicentin-1 is an essential extracellular matrix component of the dermal-epidermal and myotendinous junctions.

The extracellular matrix architecture is composed of supramolecular fibrillar networks that define tissue specific cellular microenvironments. Hemicentins (Hmcn1 and Hmcn2) are ancient and very large members (> 600 kDa) of the fibulin family, whose short members are known to guide proper morphology and functional behavior of specialized cell types predominantly in elastic tissues. However, the tissue distribution and function of Hemicentins within the cellular microenvironment of connective tissues has remained largely unknown. Performing in situ hybridization and immunofluorescence analyses, we found that mouse Hmcn1 and Hmcn2 show a complementary distribution throughout different tissues and developmental stages. In postnatal dermal-epidermal junctions (DEJ) and myotendinous junctions (MTJ), Hmcn1 is primarily produced by mesenchymal cells (fibroblasts, tenocytes), Hmcn2 by cells of epithelial origin (keratinocytes, myocytes). Hmcn1-/- mice are viable and show no overt phenotypes in tissue tensile strength and locomotion tests. However, transmission electron microscopy revealed ultrastructural basement membrane (BM) alterations at the DEJ and MTJ of Hmcn1-/- mice, pointing to a thus far unknown role of Hmcn1 for BM and connective tissue boundary integrity.

Anisotropic and age-dependent elastic material behavior of the human costal cartilage.

Compared to articular cartilage, the biomechanical properties of costal cartilage have not yet been extensively explored. The research presented addresses this problem by studying for the first time the anisotropic elastic behavior of human costal cartilage. Samples were taken from 12 male and female cadavers and unconfined compression and indentation tests were performed in mediolateral and dorsoventral direction to determine Young's Moduli EC for compression and Ei5%, Ei10% and Eimax at 5%, 10% and maximum strain for indentation. Furthermore, the crack direction of the unconfined compression samples was determined and histological samples of the cartilage tissue were examined with the picrosirius-polarization staining method. The tests revealed mean Young's Moduli of EC = 32.9 ± 17.9 MPa (N = 10), Ei5% = 11.1 ± 5.6 MPa (N = 12), Ei10% = 13.3 ± 6.3 MPa (N = 12) and Eimax = 14.6 ± 6.6 MPa (N = 12). We found that the Young's Moduli in the indentation test are clearly anisotropic with significant higher results in the mediolateral direction (all P = 0.002). In addition, a dependence of the crack direction of the compressed specimens on the load orientation was observed. Those findings were supported by the orientation of the structure of the collagen fibers determined in the histological examination. Also, a significant age-related elastic behavior of human costal cartilage could be shown with the unconfined compression test (P = 0.009) and the indentation test (P = 0.004), but no sex effect could be detected. Those results are helpful in the field of autologous grafts for rhinoplastic surgery and for the refinement of material parameters in Finite Element models e.g., for accident analyses with traumatic impact on the thorax.

Plastin 3 in health and disease: a matter of balance.

For a long time, PLS3 (plastin 3, also known as T-plastin or fimbrin) has been considered a rather inconspicuous protein, involved in F-actin-binding and -bundling. However, in recent years, a plethora of discoveries have turned PLS3 into a highly interesting protein involved in many cellular processes, signaling pathways, and diseases. PLS3 is localized on the X-chromosome, but shows sex-specific, inter-individual and tissue-specific expression variability pointing towards skewed X-inactivation. PLS3 is expressed in all solid tissues but usually not in hematopoietic cells. When escaping X-inactivation, PLS3 triggers a plethora of different types of cancers. Elevated PLS3 levels are considered a prognostic biomarker for cancer and refractory response to therapies. When it is knocked out or mutated in humans and mice, it causes osteoporosis with bone fractures; it is the only protein involved in actin dynamics responsible for osteoporosis. Instead, when PLS3 is upregulated, it acts as a highly protective SMN-independent modifier in spinal muscular atrophy (SMA). Here, it seems to counteract reduced F-actin levels by restoring impaired endocytosis and disturbed calcium homeostasis caused by reduced SMN levels. In contrast, an upregulation of PLS3 on wild-type level might cause osteoarthritis. This emphasizes that the amount of PLS3 in our cells must be precisely balanced; both too much and too little can be detrimental. Actin-dynamics, regulated by PLS3 among others, are crucial in a lot of cellular processes including endocytosis, cell migration, axonal growth, neurotransmission, translation, and others. Also, PLS3 levels influence the infection with different bacteria, mycosis, and other pathogens.

Insights to enhance the examination of tool marks in human cartilage.

This work deals with the examination of tool marks in human cartilage. We compared the effectiveness of several cleaning methods on cut marks in porcine cartilage. The method cleaning by multiple casts achieved the significantly highest scores (P = 0.02). Furthermore, we examined the grain-like elevations (dots) located on casts of cut cartilage. The results of this study suggest that the casting material forms these dots when penetrating cartilage cavities, which are areas where the strong collagen fibres leave space for the chondrocytes. We performed fixation experiments to avoid this, without success. In addition, 31 casting materials were compared regarding contrast under light-microscope and 3D tool marks scanner. Under the light-microscope, brown materials achieved significantly higher values than grey (P = 0.02) or black (P = 0.00) whereas under the 3D scanner, black materials reached higher contrast values than grey (P = 0.04) or brown (P = 0.047). To compare the accuracy and reproducibility of 6 test materials for cartilage, we used 10 knives to create cut marks that were subsequently scanned. During the alignment of the individual signals of each mark, the cross-correlation coefficients (Xmax) and lags (LXmax) were calculated. The signals of the marks in agarose were aligned with significantly fewer lags and achieved significantly higher cross-correlation coefficients compared to all tested materials (both P = 0.00). Moreover, we determined the cross-correlation coefficients (XC) for known-matches (KM) per material. Agarose achieved significantly higher values than AccuTrans®, Clear Ballistics™, and gelatine (all P = 0.00). The results of this work provide valuable insights for the forensic investigation of marks in human costal cartilage.

Expression and Localization of Thrombospondins, Plastin 3, and STIM1 in Different Cartilage Compartments of the Osteoarthritic Varus Knee.

Osteoarthritis (OA) is a multifactorial disease which is characterized by a change in the homeostasis of the extracellular matrix (ECM). The ECM is essential for the function of the articular cartilage and plays an important role in cartilage mechanotransduction. To provide a better understanding of the interaction between the ECM and the actin cytoskeleton, we investigated the localization and expression of the Ca2+-dependent proteins cartilage oligomeric matrix protein (COMP), thrombospondin-1 (TSP-1), plastin 3 (PLS3) and stromal interaction molecule 1 (STIM1). We investigated 16 patients who suffered from varus knee OA and performed a topographical analysis of the cartilage from the medial and lateral compartment of the proximal tibial plateau. In a varus knee, OA is more pronounced in the medial compared to the lateral compartment as a result of an overloading due to the malalignment. We detected a location-dependent staining of PLS3 and STIM1 in the articular cartilage tissue. The staining intensity for both proteins correlated with the degree of cartilage degeneration. The staining intensity of TSP-1 was clearly reduced in the cartilage of the more affected medial compartment, an observation that was confirmed in cartilage extracts by immunoblotting. The total amount of COMP was unchanged; however, slight changes were detected in the localization of the protein. Our results provide novel information on alterations in OA cartilage suggesting that Ca2+-dependent mechanotransduction between the ECM and the actin cytoskeleton might play an essential role in the pathomechanism of OA.

Short-term Response of Serum Cartilage Oligomeric Matrix Protein to Different Types of Impact Loading Under Normal and Artificial Gravity.

Microgravity during long-term space flights induces degeneration of articular cartilage. Artificial gravity through centrifugation combined with exercise has been suggested as a potential countermeasure for musculoskeletal degeneration. The purpose of this study was to investigate the effect of different types of impact loading under normal and artificial gravity conditions on serum concentrations of cartilage oligomeric matrix protein (COMP), a biomarker of cartilage metabolism. Fifteen healthy male adults (26 ± 4 years, 181 ± 4 cm, 77 ± 6 kg) performed four different 30-min impact loading protocols on four experimental days: jumping with artificial gravity elicited by centrifugation in a short-arm centrifuge (AGJ), jumping with artificial gravity generated by low-pressure cylinders in a sledge jump system (SJS), vertical jumping under Earth gravity (EGJ), and running under Earth gravity (RUN). Five blood samples per protocol were taken: 30 min before, immediately before, immediately after, 30 min after, and 60 min after impact loading. Serum COMP concentrations were analyzed in these samples. During the impact exercises, ground reaction forces were recorded. Peak ground reaction forces were significantly different between the three jumping protocols (p < 0.001), increasing from AGJ (14 N/kg) to SJS (22 N/kg) to EGJ (29 N/kg) but were similar in RUN (22 N/kg) compared to SJS. The serum COMP concentration was increased (p < 0.001) immediately after all loading protocols, and then decreased (p < 0.001) at 30 min post-exercise compared to immediately after the exercise. Jumping and running under Earth gravity (EGJ and RUN) resulted in a significantly higher (p < 0.05) increase of serum COMP levels 30 min after impact loading compared to the impact loading under artificial gravity (RUN +30%, EGJ +20%, AGJ +17%, and SJS +13% compared to baseline). In conclusion, both the amplitude and the number of the impacts contribute to inducing higher COMP responses and are therefore likely important factors affecting cartilage metabolism. RUN had the largest effect on serum COMP concentration, presumably due to the high number of impacts, which was 10 times higher than for the jump modalities. Future studies should aim at establishing a dose-response relationship for different types of exercise using comparable amounts of impacts.

Role of collagen XII in skin homeostasis and repair.

Skin integrity and function depends to a large extent on the composition of the extracellular matrix, which regulates tissue organization. Collagen XII is a homotrimer with short collagenous domains that confer binding to the surface of collagen I-containing fibrils and extended flexible arms, which bind to non-collagenous matrix components. Thereby, collagen XII helps to maintain collagen suprastructure and to absorb stress. Mutant or absent collagen XII leads to reduced muscle and bone strength and lax skin, whereas increased collagen XII amounts are observed in tumor stroma, scarring and fibrosis. This study aimed at uncovering in vivo mechanisms by which collagen XII may achieve these contrasting outcomes. We analyzed skin as a model tissue that contains abundant fibrils, composed of collagen I, III and V with collagen XII decorating their surface, and which is subject to mechanical stress. The impact of different collagen XII levels was investigated in collagen XII-deficient (Col12-KO) mice and in mice with collagen XII overexpression in the dermis (Col12-OE). Unchallenged skin of these mice was histologically inconspicuous, but at the ultrastructural level revealed distinct aberrations in collagen network suprastructure. Repair of excisional wounds deviated from controls in both models by delayed healing kinetics, which was, however, caused by completely different mechanisms in the two mouse lines. The disorganized matrix in Col12-KO wounds failed to properly sequester TGFß, resulting in elevated numbers of myofibroblasts. These are, however, unable to contract and remodel the collagen XII-deficient matrix. Excess of collagen XII, in contrast, promotes persistence of M1-like macrophages in the wound bed, thereby stalling the wounds in an early inflammatory stage of the repair process and delaying healing. Taken together, we demonstrate that collagen XII is a key component that assists in orchestrating proper skin matrix structure, controls growth factor availability and regulates cellular composition and function. Together, these functions are pivotal for re-establishing homeostasis after injury.

Locomotion replacement exercise cannot counteract cartilage biomarker response to 5 days of immobilization in healthy adults.

Biomarkers of cartilage metabolism are sensitive to changes in the biological and mechanical environment and can indicate early changes in cartilage homeostasis. The purpose of this study was to determine if a daily locomotion replacement program can serve as a countermeasure for changes in cartilage biomarker serum concentration caused by immobilization. Ten healthy male subjects (mean ± 1 standard deviation; age: 29.4 ± 5.9 years; body mass: 77.7 ± 4.1 kg) participated in the crossover 5-day bed rest study with three interventions: control (CON), standing (STA), and locomotion replacement training (LRT). Serum samples were taken before, during, and after bed rest. Biomarker concentrations were measured using commercial enzyme-linked immunosorbent assays. Cartilage oligomeric matrix protein (COMP) levels after 24 hours of bed rest decreased independently of the intervention (-16.8% to -9.8%) and continued to decrease until 72 hours of bed rest (minimum, -23.2% to -20.6%). LRT and STA did not affect COMP during bed rests (P = .056) but there was a strong tendency for a slower decrease with LRT (-9.4%) and STA (-11.7%) compared with CON (-16.8%). MMP-3 levels decreased within the first 24 hours of bed rest (CON: -22.3%; STA: -14.7%; LRT: -17%) without intervention effect. Both COMP and MMP-3 levels recovered to baseline levels during the 6-day recovery period. MMP-1, MMP-9, and TNF-α levels were not affected by immobilization or intervention. COMP and MMP-3 are mechano-sensitive cartilage biomarkers affected by immobilization, and simple interventions such as standing upright or LRT during bed rest cannot prevent these changes. Clinical significance: simple locomotion interventions cannot prevent cartilage biomarker change during bed rest.