Associate Professor Terhi Heino, University of Turku, Finland
Docent Lindsay Davies, Karolinska Institute, Stockholm, Sweden
Docent Jonathan Gilthorpe, University of Umeå, Sweden
Professor Sture Forsberg, University of Umeå, Sweden
Professor Mikko Lammi, University of Umeå, Sweden
Docent Jukka Häyrinen, University of Eastern Finland, Finland
Articles of the thesis
Osteoarthritis is an inflammation-related disease that progressively destroys joint cartilage. This disease causes pain and stiffness of the joints, and at advanced stages, limitations to the movement or bending of injured joints. Therefore, it often restricts daily activities and the ability to work. Currently, there is no cure to prevent its progression, although certain damaged joints, such as fingers, knees and hips, can be treated with joint replacement surgeries. However, joint replacement surgeries of larger joints are very invasive operations and the joint replacements have a limited lifetime.
Cell-based therapies could offer a way to treat cartilage injuries before the ultimate damage of osteoarthritis on articular cartilage. The development of novel treatments needs both a good knowledge of articular cartilage biology and tissue engineering methods. This thesis primarily investigates the effects of mechanical cyclic stretching, a 5% low oxygen atmosphere and the Rho-kinase inhibitor, Y-27632, on protein responses in chondrocytic human chondrosarcoma (HCS-2/8) cells. Special focus is placed on Rho-kinase inhibition, relating to its potential to promote and support extracellular matrix production in cultured chondrocytes and its role in fibroblast cells as a part of direct chemical cellular differentiation. The means to enhance the production of cartilage-specific extracellular matrix is needed for cell-based tissue engineering applications, since cultured chondrocytes quickly lose their cartilage-specific phenotype.
A mechanical 8% cyclic cell stretching at a 1 Hz frequency was used to model a stretching rhythm similar to walking. The cellular stretching relates to stresses, which are directed to chondrocytes during the mechanical load. The stretch induced changes in proteins related, e.g., to certain cytoskeletal proteins, but also in enzymes associated with protein synthesis, such as eukaryotic elongation factors 1-beta and 1-delta. Hypoxic conditions were used to model the oxygen tension present in healthy cartilage tissue. Long-term hypoxia changed relative amounts in a total of 44 proteins and induced gene expressions of aggrecan and type II collagen, in addition to chondrocyte differentiation markers S100A1 and S100B. A short-term inhibition of Rho-kinase failed to induce extracellular matrix production in fibroblasts or in HCS-2/8 cells, while its long-term exposure increased the expressions of chondrocyte-specific genes and differentiation markers, and also promoted the synthesis of sulfated glycosaminoglycans by chondrocytic cells. Interestingly, Rho kinase inhibition under hypoxic conditions produced a more effective increase in chondrocyte-specific gene expression and synthesis of extracellular matrix components by HCS-2/8 cells. The treatment induced changes in the synthesis of 101 proteins and ELISA analysis revealed a sixfold higher secretion of type II collagen compared to control cells. The secretion of sulfated glycosaminoglycans was simultaneously increased by 65.8%. Thus, Rho-kinase inhibition at low oxygen tension can be regarded as a potential way to enhance extracellular matrix production and maintain a chondrocyte phenotype in cell-based tissue engineering applications.
Piltti J, Häyrinen J, Karjalainen HM, Lammi MJ: Proteomics of chondrocytes with special reference to phosphorylation changes of proteins in stretched human chondrosarcoma cells. Biorheology 45(3-4):323-335, 2008 [Pubmed] [Full text]
Piltti J, Varjosalo M, Qu C, Häyrinen J, Lammi MJ: Rho-kinase Y27632 increases cellular proliferation and migration in human foreskin fibroblast cells. Proteomics, 15(17): 2953-2965, 2015 [Pubmed] [Full text]
Piltti J, Bygdell J, Fernandez-Echevarria C, Marcellino D, Lammi MJ: Rho-kinase inhibitor Y-27632 and hypoxia synergistically enhance chondrocytic phenotype and modify the S100 protein profile in human chondrosarcoma cells. Sci Rep 7(1): 3708, 2017 [Pubmed] [Full text]
Lammi MJ, Piltti J, Prittinen J, Qu C: Challenges in fabrication of tisue-engineered cartilage with correct cellular colonization and extracellular matrix assembly. Int J Mol Sci 19(9): 2700, 2018 [Pubmed] [Full text]
Prittinen J, Ylärinne J, Piltti J, Karhula S, Rieppo L, Ojanen P, Korhonen RK, Saarakkala S, Lammi MJ, Qu C: Effect of centrifugal force on the development of articular neocartilage with bovine primary chondrocytes. Cell Tissue Res 375(3): 629-639, 2019 [Pubmed] [Full text]
Last updated March 7, 2019