The influence of ECM on mesenchymal stromal/stem cell activity and their interactions with monocytes/macrophages
Osteoarthritis is a prevalent degenerative condition affecting synovial joints, resulting in pain, stiffness, and difficulty with movement. Current treatments mainly alleviate symptoms without effectively regenerating the lost cartilage tissue. An early surgical intervention for focal cartilage damage, known as marrow stimulation or microfracture, aims to repair this damage by promoting endogenous healing. However, the resulting repair tissue often lacks the properties of native cartilage, raising questions about the long-term effectiveness of this approach. Therefore, to enhance the outcome of this osteochondral repair approach, it is necessary to understand the biological mechanisms involved in the healing process following microfracture. Osteochondral repair is a complex interplay of cellular interactions and changing extracellular matrix (ECM) environment. This thesis aimed to extend the current understanding of the biology underpinning this process, focusing on the interaction between mesenchymal stromal/stem cells (MSCs) and monocytes/macrophages within dynamically changing ECM proteins found at the injury site. We hypothesized that this interaction plays a pivotal role in determining the outcome of tissue repair. In our investigation, we first characterised the deposition and remodelling patterns of key ECM proteins, fibrin and collagen type 1, during osteochondral injury repair in C57BL/6 mice. Our findings revealed that fibrin was the predominant ECM protein in the initial seven days post-injury, which was subsequently gradually replaced by collagen type 1. At eight weeks post-injury, the tissue repair outcome at the articular surface was found to contain collagen type 1, perhaps representing an immature form of hyaline cartilage. Subsequent *in vitro* studies, using 3D ECM models constructed from fibrin and collagen type 1, demonstrated that these environments significantly modulate MSC morphology, proliferation, migration, and immunomodulatory activity. Notably, MSCs within these 3D environments exhibit differential responses to pro-inflammatory stimuli, with the ECM potentially acting as a reservoir for secreted cytokines and growth factors, orchestrating cellular activities over time. Three molecular mechanisms, the TNFα/NFkB pathway, TNFα/JNK/AP1 pathway, and the potential involvement of ROCK, were identified as specific effectors of MSC immunomodulatory activity within these environments. Further investigations into the crosstalk between MSCs and monocytes in controlled 3D ECM environments revealed a distinct M2 macrophage phenotype, characterized as CD206+ MerTK- CD163- CD209-. This subpopulation displayed enhanced expression of *IL10*, IL6, and IL8. Moreover, conditioned media from these co-cultures influenced chondrocyte migration and chondrogenesis, with TGFβ1 playing a pivotal role in these observations. In conclusion, this thesis underscores the profound influence of the ECM on cellular interactions during osteochondral repair. The insights gained pave the way for innovative therapeutic strategies, potentially enhancing tissue repair outcomes and offering improved treatments for conditions like osteoarthritis.
Extracellular matrix, macrophages, mesenchymal stromal/stem cells, monocytes, Osteoarthritis, osteochondral repair
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