engleski

Optimization of conditions for in vitro three-dimensional cartilage growth

Articular cartilage is a poorly vascularized and innervated tissue that shows no capacity for effective spontaneous regeneration in cases of damage and injury, which represents a major health problem and unmet medical need. Common methods of the treatment and therapy have proved to be ineffective. Tissue engineering, as a new important field of regenerative medicine, emerges as potential effective alternative. Chondrogenesis, the process by which cartilage is formed, actually represents the consequence of several steps directed by signaling molecules, receptors, transcription factors, cells’ interaction with ECM and other environmental factors. In this respect, the aim of our study was to optimize conditions for 3D in vitro chondrogenesis in order to produce functional cartilage implants that could be used in clinical practice to cure some specific cartilage defects. The method of optimization includs: i) cell type ; ii) source of dexamethasone ; and iii) percentage of oxygen. In the experiment three different cell culture types were used: chondrocytes, human mesenchymal stem cells from bone marrow (hMSC) and combination of chondrocytes and hMSC in 2:1 ratio. Cells were grown in 3D culture, incorporated in a peptide hydrogel RADA (BD PuraMatrix Peptide Hydrogel). In order to induce chondrogenesis, cells were put in a differentiation medium containing ascorbic acid-2-phosphate, L- proline, ITS, TGFβ-1 and dexamethasone. Dexamethasone was added in a differentiation medium or it was incorporated with cells within a peptide hydrogel. In order to demonstrate the effect of oxygen level on the efficiency of chondrogenic differentiation, cells were grown in normoxic (20% O2) and hypoxic conditions (5% O2). Expression levels of two important cartilage marker genes, SOX9 and agreccan, were evaluated by quantitative PCR in samples on day10 and day21 after induction of chondrogenesis. Our results provide important insights into effects of how different cell type, dexamethasone source and oxygen level affect efficiency of in vitro chondrogenesis.

cartilage ; chondrogenesis ; chondrocytes ; human mesenchymal stem cells ; peptide hydrogel

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