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Title: Dynamic Mechanical Stimulation for Mesenchymal Stem Cell Chondrogenesis in An Elastomeric Scaffold
Keywords: Chondrogenesis, compression, shearing, pathways
Issue Date: 22-Aug-2014
Citation: ZHANG TIANTING (2014-08-22). Dynamic Mechanical Stimulation for Mesenchymal Stem Cell Chondrogenesis in An Elastomeric Scaffold. ScholarBank@NUS Repository.
Abstract: Background: Mesenchymal stem cell (MSC) based cartilage tissue engineering for treating articular lesion is of particular interest due to the multipotency for effective chondrogenic differentiation. The various applications of three dimensional scaffolds and mechanical stimulations aim to promote MSC chondrogenesis in every aspect from cell attachment, proliferation to extracellular matrix (ECM) deposition and mechanical properties. Hypothesis: The general hypothesis of this thesis is that deferral dynamic mechanical stimulation is able to enhance chondrogenesis, suppress hypertrophy and potentially facilitate zonal distribution in the MSC constructs supported with stepwise upgraded elastomeric poly L-lactide-co-e-caprolactone (PLCL) scaffolds. It was broken down into three sections of investigation. Methods: In vitro studies were conducted on MSC-seeded scaffolds. Chondrogenic culture and mechanical stimulation of compression and dual-axis loading were applied to constructs. The porous PLCL, unilayered PLCL/chitosan and bilayered PLCL/chitosan scaffolds were characterized using SEM, FTIR/TGA and mechanical tests. The cell-matrix constructs were evaluated by histological analysis, chondrogenic/hypertrophic/zonal mRNAs expression, protein synthesis level and mechanical strength analysis. Mechanism of mechanotransduction was studied through assessing the regulation of pathway relevant molecules in TGF-?/SMAD and integrin ?1 signaling. Results: In the first section, chitosan coating on PLCL scaffold increased hydrophilicity, which further promoted cell spreading, attachment, distribution and condensation. MSC-seeded PLCL/chitosan constructs showed increasing expression of collagen type II (COL II) and aggrecan (AGCAN), as well as higher mechanical strength. In the second study, deferral dynamic compression enhanced COL II and AGCAN deposition and suppressed collagen type X (COL X), matrix metallopeptidase 13 (MMP13), alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2) expression. A further investigation on TGF-?/SMAD and integrin ?1 pathways showed compression promoted phosphorylation of SMAD2/3, but down-regulated phosphorylation of SMAD1/5/8, focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). These molecular modulations were confirmed by ALK5 and integrin ?1 inhibition. In the last results chapter, the application of deferral dual-axis (DA) loading to MSC-seeded bilayered PLCL/chitosan constructs showed a decrease in AGCAN mRNA expression and an increase in COL II staining in the layer with small pore (SP). Subsequent mRNA analysis of zonal markers ? collagen type I (COL I) and proteoglycan 4 (PRG4) exhibited increased expression in SP layer under DA loading, indicating the occurrence of ECM zonal deposition. Conclusion: Deferral dynamic compression enhanced MSC chondrogenesis in hydrophilic unilayered PLCL/chitosan scaffold, and inhibited hypertrophic development. The mechanotransduction of compression initiated from transducing extracellular physical loading into intracellular biochemical signals through integrin ?1 pathway. Then crosstalk between TGF-?/SMAD and integrin ?1siganling leads to chondrogenic enhancement and hypertrophic suppression through the antagonizing roles of TGF-?/Activin/Nodal and BMP/GDP branches. The further application of deferral dual-axis loading to bilayered PLCL/chitosan MSC constructs revealed potential effect on zonal cartilaginous ECM constituents formation.
Appears in Collections:Ph.D Theses (Open)

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