Direct comparison of non-osteoarthritic and osteoarthritic synovial fluid-induced intracellular chondrocyte signaling and phenotype changes.

ObjectiveSince the joint microenvironment and tissue homeostasis are highly dependent on synovial fluid, we aimed to compare the essential chondrocyte signaling signatures of non-osteoarthritic vs end-stage osteoarthritic knee synovial fluid. Moreover, we determined the phenotypic consequence of the distinct signaling patterns on articular chondrocytes.MethodsProtein profiling of synovial fluid was performed using antibody arrays. Chondrocyte signaling and phenotypic changes induced by non-osteoarthritic and osteoarthritic synovial fluid were analyzed using a phospho-kinase array, luciferase-based transcription factor activity assays, and RT-qPCR. The origin of osteoarthritic synovial fluid signaling was evaluated by comparing the signaling responses of conditioned media from cartilage, synovium, infrapatellar fat pad and meniscus. Osteoarthritic synovial fluid induced pathway-phenotype relationships were evaluated using pharmacological inhibitors.ResultsCompared to non-osteoarthritic synovial fluid, osteoarthritic synovial fluid was enriched in cytokines, chemokines and growth factors that provoked differential MAPK, AKT, NFκB and cell cycle signaling in chondrocytes. Functional pathway analysis confirmed increased activity of these signaling events upon osteoarthritic synovial fluid stimulation. Tissue secretomes of osteoarthritic cartilage, synovium, infrapatellar fat pad and meniscus activated several inflammatory signaling routes. Furthermore, the distinct pathway signatures of osteoarthritic synovial fluid led to accelerated chondrocyte dedifferentiation via MAPK/ERK signaling, increased chondrocyte fibrosis through MAPK/JNK and PI3K/AKT activation, an elevated inflammatory response mediated by cPKC/NFκB, production of extracellular matrix-degrading enzymes by MAPK/p38 and PI3K/AKT routes, and enabling of chondrocyte proliferation.ConclusionThis study provides the first mechanistic comparison between non-osteoarthritic and osteoarthritic synovial fluid, highlighting MAPKs, cPKC/NFκB and PI3K/AKT as crucial OA-associated intracellular signaling routes

Numerical methods for the design and description of in vitro expansion processes of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction or inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hMSC-based therapies, in vitro expansion is necessary prior to the clinical application in order to achieve the required cell numbers. Safe, reproducible, and economic in vitro expansion of hMSCs for autologous and allogeneic therapies can be problematic because the cell material is restricted and the cells are sensitive to environmental changes. It is beneficial to collect detailed information on the hydrodynamic conditions and cell growth behavior in a bioreactor system, in order to develop a so called “Digital Twin” of the cultivation system and expansion process. Numerical methods, such as Computational Fluid Dynamics (CFD) which has become widely used in the biotech industry for studying local characteristics within bioreactors or kinetic growth modelling, provide possible solutions for such tasks. In this review, we will present the current state-of-the-art for the in vitro expansion of hMSCs. Different numerical tools, including numerical fluid flow simulations and cell growth modelling approaches for hMSCs, will be presented. In addition, a case study demonstrating the applicability of CFD and kinetic growth modelling for the development of an microcarrier-based hMSC process will be shown

Evaluation of impaired growth plate development of long bones in skeletally immature mice by antirheumatic agents

Restriction of physical growth and development is a known problem in patients with juvenile idiopathic arthritis (JIA). However, the effect of medical treatment for JIA on skeletal growth in affected children has not been properly investigated. We, therefore, hypothesize that naproxen and methotrexate (MTX) affect endochondral ossification and will lead to reduced skeletal development. Treatment of ATDC5 cells, an in vitro model for endochondral ossification, with naproxen or MTX resulted in increased chondrogenic but decreased hypertrophic differentiation. In vivo, healthy growing C57BL/6 mice were treated with naproxen, MTX, or placebo for 10 weeks. At 15 weeks postnatal, both the length of the tibia and the length of the femur were significantly reduced in the naproxen- and MTX-treated mice compared to their controls. Growth plate analysis revealed a significantly thicker proliferative zone, while the hypertrophic zone was significantly thinner in both experimental groups compared to their controls, comparable to the in vitro results. Micro-computed tomography analysis of the subchondral bone region directly below the growth disc showed significantly altered bone microarchitecture in naproxen and MTX groups. In addition, the involvement of the PTHrP-Ihh loop in naproxen- and MTX-treated cells was shown. Overall, these results demonstrate that naproxen and MTX have a profound effect on endochondral ossification during growth plate development, abnormal subchondral bone morphology, and reduced bone length. A better understanding of how medication influences the development of the growth plate will improve understanding of endochondral ossification and reveal possibilities to improve the treatment of pediatric patients