Abstract
Introduction: The knee meniscus is prone to injury and has limited intrinsic healing potential despite surgical repair. Methods to enhance fibrochondrocyte function and augment meniscal repair would be invaluable in the treatment of meniscal injuries. Ultraviolet Ozone (UVO) modified surfaces have been characterised chemically and topographically. These surfaces have been shown to promote the function of certain cell types. This study investigated the attachment, proliferation and extracellular matrix production of fibrochondrocytes cultured on UVO modified polystyrene surfaces. Interest was paid to the integrins, a group of transmembrane extracellular matrix attachment glycoproteins. In particular the subunits alpha2 and alpha5, as they specifically bind to the ligands Collagen Type I and Fibronectin, major components in the human meniscus.
Methods: Tissue samples from adult human medial meniscal tears were obtained at knee arthroscopy. Fibrochondrocytes were isolated by standard cell culture techniques and cultured to 100% confluence before seeding onto UVO modified polystyrene surfaces. The untreated polystyrene surfaces of culture dishes were oxidatively modified with an ultraviolet ozone treater. The response of fibrochondrocytes to various surface oxygen concentrations was investigated. Untreated, hydrophobic surfaces acted as controls. Images of cells in culture were obtained with a Leica digital camera mounted on a microscope. Cells were counted at 24, 48, 72 and 96 hours. After 48 and 96 hours of culture standard wet transfer Western Blots were undertaken using antibodies to the alpha2 and aalpha5 integrin subunits (Santa Cruz Biotechnology). To evaluate potential extracellular matrix production total protein assays were undertaken at 1, 2, 3, 4, 6, 8 and 10 days of culture (Bio-Rad Laboratories).
Results: Fibrochondrocytes attached preferentially to the UVO treated surfaces. They proliferated steadily until they reached confluence at 96 hours. Western Blot analysis showed the integrin subunit a5 to be present in the cell lysate after both 48 and 96 hours of culture. The a2 subunit was not detected at these times. There was no increase in total protein concentration on surfaces after fibrochondrocytes had reached confluence. Discussion: UVO modified surfaces promote the attachment and proliferation of human fibrochondrocytes. The alpha2 subunit was not detected in the cell lysate of these surfaces after culture for 96 hours. Whether this is due to defective or absent Collagen Type I at this stage of culture remains to be answered. The presence of the alpha5 subunit suggests that Fibronectin may be involved in the process of fibrochondrocyte attachment to UVO modified polystyrene surfaces. It is suggested that there is little or no extracellular matrix production after 4 days as there is no increase in total surface protein concentration after confluence is reached. The increase in total surface protein concentration up to this point most likely reflects cell proliferation.
Conclusion: Ultraviolet Ozone modified surfaces enhance certain aspects of fibrochondrocyte function and therefore have a potential role in the development of novel therapies for meniscal repair.
Correspondence should be addressed to Dr Carlos Wigderowitz, Honorary Secretary of BORS, Division of Surgery & Oncology, Section of Orthopaedic & Trauma Surgery, Ninewells Hospital & Medical School Tort Centre, Dundee, DD1 9SY.
