Induction of chondrogenesis from MSCs by BMP7 and TGFB1 in low oxygen tension: expression of superficial zone protein

Project (M.A., Biological Sciences (Stem Cell))--California State University, Sacramento, 2012.Osteoarthritis (OA) is the most common form of arthritis, afflicting 12.1% of the United States population. OA is characterized by the degeneration of articular cartilage, thus its synonym degenerative arthritis. Articulating ends of bone found within a synovial joint are lined with articular cartilage. Articular cartilage imparts extremely important mechanical properties that give the joint's ability to function properly. Even under high loads, articular cartilage slide together creating near frictionless motion comparable to ice-on-ice. Due to the avascular nature of articular cartilage, the oxygen tension of the tissue is as low as 1%, and makes delivering pharmaceuticals via the blood stream nearly impossible. OA patients have limited treatment options, mainly focusing on treating inflammation and pain. My goal is to differentiate human bone marrow derived mesenchymal stem cells (hBM-MSCs) into articular cartilage with morphogens BMP-7 and TGF??1. Differentiating stem cells into chondrocytes in hypoxic conditions has been shown to increase chondrogenesis. I am comparing the expression of superficial zone protein (SZP) by MSC differentiated at atmospheric (27% O2) and hypoxic (3% O2) oxygen concentrations. The degree of differentiation of MSCs into articular cartilage will be assessed on the pellets expression of articular cartilage proteins (collagen, aggrecan, superficial zone protein etc.) and glycosaminoglycans. Differentiated MSCs are evaluated through histologically using Toluidine Blue staining for glycosaminoglycans, and immunohistochemically for the expression of superficial zone protein. The mRNA expression of Aggrecan, collagen I and superficial zone protein of the cells in pellet cell culture is quantified using qPCR.\ud Results show no increase of chondrogenesis or SZP expressed in MSC pellets differentiated in hypoxic environment. Toluidine blue stain shows no increase glycosaminoglycans, a key element in articular cartilage matrixes. Although western blot showed more SZP secreted in the media of pellets in low oxygen environment, both immunohistochemistry and qPCR show a decrease of SZP in pellets differentiated in hypoxic environment. Other chondrogenic markers such as Sox9 and aggrecan were also shown to be decreased in the hypoxia differentiated pellets.Biological Sciences (Stem Cell