Anja Niehoff

Deformational behaviour of knee cartilage and changes in serum cartilage oligomeric matrix protein (COMP) after running and drop landing

OBJECTIVE To investigate (1) the effect of running and drop landing interventions on knee cartilage deformation and serum cartilage oligomeric matrix protein (COMP) concentration and (2) if the changes in cartilage volume correlate with the changes in serum COMP level. METHODS Knee joint cartilage volume and thickness were determined using magnetic resonance imaging (MRI) as well as COMP concentration from serum samples before and after in vivo loading of 14 healthy adults (seven male and seven female). Participants performed different loading interventions of 30 min duration on three different days: (1) 100 vertical drop landings from a 73 cm high platform, (2) running at a velocity of 2.2m/s (3.96 km), and (3) resting on a chair. Blood samples were taken immediately before, immediately after and 0.5h, 1h, 2h and 3h post intervention. Pre- and post-loading coronal and axial gradient echo MR images with fat suppression were used to determine the patellar, tibial and femoral cartilage deformation. RESULTS Serum COMP levels increased immediately after the running (+30.7%, pre: 7.3U/l, 95% confidence interval (CI): 5.6, 8.9, post: 9.1U/l, 95% CI: 7.2, 11.0, P=0.001) and after drop landing intervention (+32.3%, pre: 6.8U/l, 95% CI: 5.3, 8.4; post: 8.9U/l, 95% CI: 6.8, 10.9, P=0.001). Cartilage deformation was more pronounced after running compared to drop landing intervention, with being significant (volume: P=0.002 and thickness: P=0.001) only in the lateral tibia. We found a significant correlation (r(2)=0.599, P=0.001) between changes in serum COMP (%) and in cartilage volume (%) after the drop landing intervention, but not after running. CONCLUSIONS In vivo exercise interventions differentially regulate serum COMP concentrations and knee cartilage deformations. The relation between changes in COMP and in cartilage volume seems to depend on both mechanical and biochemical factors.

Ablation of collagen IX and COMP disrupts epiphyseal cartilage architecture.

Chondrodysplasias are a genetically heterogeneous group of skeletal disorders. Mutations in genes coding for cartilage oligomeric matrix protein (COMP), collagen IX and matrilin-3 have been described to cause the autosomal dominantly inherited form of multiple epiphyseal dysplasia (MED). Even though there is clear evidence that these cartilage matrix proteins interact with each other, their exact functions in matrix organisation and bone development still need to be elucidated. We generated a mouse model lacking both collagen IX and COMP to study the potential complementary role of these proteins in skeletal development. Mice deficient in both proteins exhibit shortened and widened long bones as well as an altered bone structure. They display severe growth plate abnormalities with large hypocellular areas in the central parts of the tibia. In addition, chondrocytes in the proliferative and hypertrophic zones do not show their typical columnar arrangement. These phenotypical traits were not observed in mice deficient only in COMP, while mice lacking only collagen IX showed similar growth plate disturbances and shorter and wider tibiae. The contribution of COMP to the phenotype of mice deficient in both collagen IX and COMP appears minor, even though clear differences in the deposition of matrilin-3 were detected.

Deficiency of annexins A5 and A6 induces complex changes in the transcriptome of growth plate cartilage but does not inhibit the induction of mineralization

Initiation of mineralization during endochondral ossification is a multistep process and has been assumed to correlate with specific interactions of annexins A5 and A6 and collagens. However, skeletal development appears to be normal in mice deficient for either A5 or A6, and the highly conserved structures led to the assumption that A5 and A6 may fulfill redundant functions. We have now generated mice deficient of both proteins. These mice were viable and fertile and showed no obvious abnormalities. Assessment of skeletal elements using histologic, ultrastructural, and peripheral quantitative computed tomographic methods revealed that mineralization and development of the skeleton were not significantly affected in mutant mice. Otherwise, global gene expression analysis showed subtle changes at the transcriptome level of genes involved in cell growth and intermediate metabolism. These results indicate that annexins A5 and A6 may not represent the essential annexins that promote mineralization in vivo.

Depletion of annexin A5, annexin A6, and collagen X causes no gross changes in matrix vesicle–mediated mineralization, but lack of collagen X affects hematopoiesis and the Th1/Th2 response

Numerous biochemical studies have pointed to an essential role of annexin A5 (AnxA5), annexin A6 (AnxA6), and collagen X in matrix vesicle–mediated biomineralization during endochondral ossification and in osteoarthritis. By binding to the extracellular matrix protein collagen X and matrix vesicles, annexins were proposed to anchor matrix vesicles in the extracellular space of hypertrophic chondrocytes to initiate the calcification of cartilage. However, mineralization appears to be normal in mice lacking AnxA5 and AnxA6, whereas collagen X–deficient mice show only subtle alterations in the growth plate organization. We hypothesized that the simultaneous lack of AnxA5, AnxA6, and collagen X in vivo induces more pronounced changes in the growth plate development and the initiation of mineralization. In this study, we generated and analyzed mice deficient for AnxA5, AnxA6, and collagen X. Surprisingly, mice were viable, fertile, and showed no obvious abnormalities. Assessment of growth plate development indicated that the hypertrophic zone was expanded in Col10a1−/− and AnxA5−/−AnxA6−/−Col10a1−/− newborns, whereas endochondral ossification and mineralization were not affected in 13‐day‐ and 1‐month‐old mutants. In peripheral quantitative computed tomography, no changes in the degree of biomineralization were found in femora of 1‐month‐ and 1‐year‐old mutants even though the diaphyseal circumference was reduced in Col10a1−/− and AnxA5−/−AnxA6−/−Col10a1−/− mice. The percentage of naive immature IgM+/IgM+ B cells and peripheral T‐helper cells were increased in Col10a1−/− and AnxA5−/−AnxA6−/−Col10a1−/− mutants, and activated splenic T cells isolated from Col10a1−/− mice secreted elevated levels of IL‐4 and GM‐CSF. Hence, collagen X is needed for hematopoiesis during endochondral ossification and for the immune response, but the interaction of annexin A5, annexin A6, and collagen X is not essential for physiological calcification of growth plate cartilage. Therefore, annexins and collagen X may rather fulfill functions in growth plate cartilage not directly linked to the mineralization process.

Effect of different running modes on the morphological, biochemical, and mechanical properties of articular cartilage.

Mechanical loading plays an important role not solely in cartilage development, but also in cartilage degeneration. Its adaptation behavior to mechanical loading has not been clearly delineated. The aim of the study was to examine the effect of different running modes (with different muscle contraction types) on morphological, biochemical, and mechanical properties of articular cartilage in the knee of growing rats. Thirty‐six female Sprague–Dawley rats were randomly assigned into a nonactive age‐matched control (AMC), level (LEVEL), and 20° downhill (DOWN) running group (n = 12 each). Running groups were trained on a treadmill for 30 min/day, 5 days/week for 6 weeks. Immunohistochemical staining and analysis of expression for collagen II, collagen IX, cartilage oligomeric matrix protein (COMP), and matrilin‐3, histomorphometry of femoral cartilage height and femoral COMP staining height, and indentation testing of tibial articular cartilage were performed. Rats subjected to downhill running showed a significantly (P = 0.015) higher COMP staining height and a tendentially (P = 0.084) higher cartilage height in the high‐weight bearing area of femoral articular cartilage. Cartilage thickness, mechanical properties, and expression of cartilage network proteins in tibial cartilage remained unaffected by different running modes. Our data suggest that joint loading induced by eccentric muscle contractions during downhill running may lead to a site‐specific adaptation.

Effect of exercise on bone and articular cartilage in heterozygous manganese superoxide dismutase (SOD2) deficient mice

Abstract Reactive oxygen species (ROS) are involved in both bone and cartilage physiology and play an important role in the pathogenesis of osteoporosis and osteoarthritis. The present study investigated the effect of running exercise on bone and cartilage in heterozygous manganese superoxide dismutase (SOD2)-deficient mice. It was hypothesized that exercise might induce an increased production of ROS in these tissues. Heterozygous SOD2-deficient mice should exhibit an impaired capability to compensate, resulting in an increased oxidative stress in cartilage and bone. Thirteen female wild type and 20 SOD2+/− mice (aged 16 weeks) were randomly assigned to a non-active wild type (SOD2+/+Con, n = 7), a trained wild type (SOD2+/+Run, n = 6), a non-active SOD2+/− (SOD2+/−Con, n = 9) and a trained SOD2+/− (SOD2+/−Run, n = 11) group. Training groups underwent running exercise on a treadmill for 8 weeks. In SOD2+/− mice elevated levels of 15-F2t-isoprostane and nitrotyrosine were detected in bone and articular cartilage compared to wild type littermates. In osteocytes the elevated levels of these molecules were found to be reduced after exercise while in chondrocytes they were increased by aerobic running exercise. The observed changes in oxidative and nitrosative stress did neither affect morphological, structural nor mechanical properties of both tissues. These results demonstrate that exercise might protect bone against oxidative stress in heterozygous SOD2-deficient mice.

Ultrastructural immunolocalization of cartilage oligomeric matrix protein, thrombospondin-4, and collagen fibril size in rodent achilles tendon in relation to exercise.

Fourteen 3-week-old Sprague-Dawley rats were housed in pairs in standard cages (5 controls) and in individual cages with a running wheel. Four of these rats had run 27–36 km/week (low training—LT) and 5 had run 56–92 km/week (high training—HT). After 4 weeks, the rats were euthanized and Achilles tendons were fixed for electron microscopy. The ultrastructural distribution of cartilage oligomeric matrix protein (COMP) and thrombospondin (TSP)-4 and collagen fibril thickness in two different extracellular compartments were studied. The immunolabeling of COMP decreased with longer running distance and was significantly lower in both the pericellular (p = 0.009) and interterritorial (p = 0.03) compartments of the HT rats compared with the controls. TSP-4 immunolabeling was higher in the pericellular compared with the interterritorial compartments in all rats (p = 0.013) but was not correlated with COMP immunolabeling. No alterations in collagen fibril size were found in relation to running; however, the gold markers representing COMP and TSP-4 were mostly found at the dark bands, representing the gap region of the fibril.

Doxycycline‐Induced Expression of Transgenic Human Tumor Necrosis Factor α in Adult Mice Results in Psoriasis‐like Arthritis

Objective To generate doxycycline-inducible human tumor necrosis factor α (TNFα)–transgenic mice to overcome a major disadvantage of existing transgenic mice with constitutive expression of TNFα, which is the limitation in crossing them with various knockout or transgenic mice. Methods A transgenic mouse line that expresses the human TNFα cytokine exclusively after doxycycline administration was generated and analyzed for the onset of diseases. Results Doxycycline-inducible human TNFα–transgenic mice developed an inflammatory arthritis– and psoriasis-like phenotype, with fore and hind paws being prominently affected. The formation of “sausage digits” with characteristic involvement of the distal interphalangeal joints and nail malformation was observed. Synovial hyperplasia, enthesitis, cartilage and bone alterations, formation of pannus tissue, and inflammation of the skin epidermis and nail matrix appeared as early as 1 week after the treatment of mice with doxycycline and became aggravated over time. The abrogation of human TNFα expression by the removal of doxycycline 6 weeks after beginning stimulation resulted in fast resolution of the most advanced macroscopic and histologic disorders, and 3–6 weeks later, only minimal signs of disease were visible. Conclusion Upon doxycycline administration, the doxycycline-inducible human TNFα–transgenic mouse displays the major features of inflammatory arthritis. It represents a unique animal model for studying the molecular mechanisms of arthritis, especially the early phases of disease genesis and tissue remodeling steps upon abrogation of TNFα expression. Furthermore, unlimited crossing of doxycycline-inducible human TNFα–transgenic mice with various knockout or transgenic mice opens new possibilities for unraveling the role of various signaling molecules acting in concert with TNFα.

Biological tissue response to impact like mechanical loading

The possible association between impact like mechanical loading and the response of biological tissue to single and repetitive impact applications in a short- and a long-term perspective is discussed on the basis of a critical review of the literature and an intervention study conducted with an athletic and a non-athletic population. The critical analysis of impact type loadings and their effect on bone and cartilage led to a more precise understanding of human impact loading. ‘Low frequency’ or ‘bounce’ type loading and ‘impact’ type loading were differentiated and used for the dedicated intervention study. The experimental results provide evidence on two complete different loading regimes related to different articular cartilage responses. The cartilage response to loading protocols at different frequencies demonstrated the higher cartilage deformation when ‘low frequency’ or ‘bouncing’ type loading than ‘high frequency’ or ‘impact’ loading was applied. The most affected cartilage plates were the patella and the tibial compartment. The study demonstrated that in vivo knee joint cartilage deformation differs following mechanical loading interventions with different amplitudes and frequencies of the forces applied. No effect was found related to different subjects representing a non-athletic and an athletic population. Cartilage volume showed no potential to adapt in the long term to mechanical and especially impact type loading.

Growth-related structural, biochemical, and mechanical properties of the functional bone-cartilage unit.

Articular cartilage and subchondral bone act together, forming a unit as a weight‐bearing loading‐transmitting surface. A close interaction between both structures has been implicated during joint cartilage degeneration, but their coupling during normal growth and development is insufficiently understood. The purpose of the present study was to examine growth‐related changes of cartilage mechanical properties and to relate these changes to alterations in cartilage biochemical composition and subchondral bone structure. Tibiae and femora of both hindlimbs from 7‐ and 13‐week‐old (each n = 12) female Sprague‐Dawley rats were harvested. Samples were processed for structural, biochemical and mechanical analyses. Immunohistochemical staining and protein expression analyses of collagen II, collagen IX, COMP and matrilin‐3, histomorphometry of cartilage thickness and COMP staining height were performed. Furthermore, mechanical testing of articular cartilage and micro‐CT analysis of subchondral bone was conducted. Growth decreased cartilage thickness, paralleled by a functional condensation of the underlying subchondral bone due to enchondral ossification. Cartilage mechanical properties seem to be rather influenced by growth‐related changes in the assembly of major ECM proteins such as collagen II, collagen IX and matrilin‐3 than by growth‐related alterations in its underlying subchondral bone structure. Importantly, the present study provides a first insight into the growth‐related structural, biochemical and mechanical interaction of articular cartilage and subchondral bone. Finally, these data contribute to the general knowledge about the cooperation between the articular cartilage and subchondral bone.