Modulation of Cellular Behaviors and Phenotypic Responses of Human Mesenchymal Cells on Biomaterials
Date Issued
2010
Date
2010
Author(s)
Lee, Yu-Tsang
Abstract
Osteogenesis and Osteoarthritis are two big issues in orthopaedic diseases. The main purpose for this study is to understand how to modulate cellular behaviors and phenotypic responses of human mesenchymal cells on different biomaterials. The cells are isolated primarily from human bone marrow and connective tissues, including synovium and cartilage of knee joints.
Human mesenchymal stem cells (hMSCs) have great potential to differentiate to lineages of mesenchymal tissues. Calcium phosphate (CaP) apatite, the main inorganic constituent of mammalian bone tissues, is believed to support hMSCs growth and osteogenic differentiation. Chitosan, a deacetylated derivative of chitin, is a versatile biopolymer to offer broad possibilities for cell-based tissue engineering. In the first part, we have applied a simple and quick method to prepare micro- and nano-scale of calcium phosphate (CaP) crystals mixed in chitosan membranes. The different concentrations of aqueous CaP suspension were mixed with chitosan in acetic acid solution and chitosan/calcium phosphate (C/CaP) films were fabricated by the solvent-casting method. The hMSCs behaviors including cell spreading, proliferation and osteogenic differentiation were examined. In basal culture medium, the addition of CaP in chitosan films could promote the proliferation of hMSCs. The films with nano-crystalline CaP significantly improved cell proliferation. In osteogenic medium, the increased alkaline phosphatase (ALP) level showed the process of osteogenic differentiation of hMSCs on the C/CaP films. The hMSCs on discrete micro-crystalline CaP films revealed higher ALP level. These results demonstrate that the crystallinity and topography of CaP apatite on chitosan membrane scaffolds modulates the behaviors of hMSCs.
Intra-articular injection of hyaluronan (hyaluronic acid; HA) is a common way to treat knee osteoarthritis (OA). This treatment can not only maintain the viscoelastic properties of knee but also release the OA pain. However, the exact molecular mechanism is still unknown. In the second part, human synovial cells were stimulated with HA (Sigma) and Hylan (Synvisc) for 24 hours. The human synovial cells were isolated from synovium tissue of advanced-staged osteoarthritic knee. Real-time polymerase chain reaction (real-time PCR) was used to detect the alteration of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) gene expression, which were specific genes related to pathogenesis of OA knees. The gene expressions of matrix-related proteins, collagen I and collagen II, were also studied. Our results illustrated that both HA and Hylan might not cause cytotoxicity or apoptosis in serum deprivation environment. For synovial cells, the cell shapes were not changed after HA and Hylan stimulation for 24 hours. The gene expressions of TGF-β1 and VEGF were significantly increased at the concentration of 0.1mg/ml HA and 0.1mg/ml Hylan, respectively. The synovial cells with treatment of 0.1mg/ml Hylan decreased the CTGF gene expression (0.66-fold) and VEGF (0.78-fold) compared to 0.1mg/ml HA. The type I collagen expressed significantly higher as treated with 0.1mg/ml HA and 0.1mg/ml Hylan.
We further isolated human cartilage cells from healthy cartilage of knee. The cartilage cells were treated with three kinds of HA, including Sigma HA, Synvisc Hylan and Artz HA, with 0.1 mg/ml and 0.01mg/ml under serum deprivation condition for 24 hours. With treatment of Synvisc Hylan, the gene expressions of CTGF, TGF-β1,VEGF, collagne I and collage II increased in 0.1mg/ml compared with 0.01mg/ml. However, the gene expression of CTGF, TGF-β1, VEGF, collagne I and collage II decreased in 0.1mg/ml relative to 0.01mg/ml with the treatment of Artz HA. Under the condition of 0.1mg/ml, Artz HA decreased the gene expressions of CTGF(0.8-fold), TGF-β1(0.8-fold) and VEGF(0.5-fold) as compared to Synvisc Hylan. Synvsic Hylan increased the gene expressions of collagen I (1.7-fold) and collagen II (4.9-fold) as compared to Artz HA. As a result, the profile of osteoarthritis-related factors of CTGF, TGF-β1 and VEGF and matrix proteins of collagen I and II might provide the rational mechanism for the therapeutic effects of hyaluronic acid on OA knees.
Subjects
mesenchymal stem cells
synovial cells
cartilage cells
nano-scale calcium phosphate
hyaluronic acid
growth factor
biomaterials
phenotype response
SDGs
Type
thesis
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