GLYCOSAMINOGLYCANS CONTENT AND TYPE II COLLAGEN LOCALIZATION IN CHONDROGENIC DIFFERENTIATION OF ADIPOSE-DERIVED MESENCHYMAL STEM CELLS INDUCED BY L-ASCORBIC ACID 2-PHOSPHATE

• Main Author: Luh Wisma Eka Yanti - Nim: 10613006, Ni
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29648
• Institution: Institut Teknologi Bandung

Articular cartilage is a type of hyaline cartilage and numerously found in diarthrodial joints. Damage in articular cartilage leading to several disorders and becomes difficult to maintain because of its limited capacity of self-repair. In vitro chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADSC) is one of alternatives to overcome the relative high amounts of chondrocytes needed in cell-based therapy. Based on previous study, L-Ascorbic Acid 2-Phosphate (LAA) has already known having a potency in the differentiation of Mesenchymal Stem Cells (MSC) to chondrocyte. Glycosaminoglycans (GAG) and type II Collagen (Coll2) are the major components of cartilage extracellular matrix which are synthesized and maintained by mature chondrocytes and used as chondrogenic markers. The objective of this study to evaluate the potency of LAA in chondrogenic differentiation of ADSC through quantification of GAG contents and Coll2 localization. Fourth passage (P4) of ADSC was characterized using flow cytometry and cultured in media containing various concentrations of LAA (0, 25, 50, 100 &#956;g/mL) for 2, 3 and 4 weeks. Coll2 localization analysis was performed by immunocytochemistry (ICC) method and visualized by confocal microscope after 2 weeks of culture, semiquantitative analysis of Coll2 localization performed using ImageJ software. The quantification of GAGs was performed by Alcian Blue staining method and the absorbance was measured at 655 nm after 3 and 4 weeks of culture. Calcium deposit quantification was performed by Alizarin Red S staining method and the absorbance was measured at 415 after 4 weeks of culture to observe the hypertrophic chondrocytes. ADSC P4 showed positive result against MSC markers such as CD73, CD90, and CD105 and negative result against CD45. The localization of Coll2 was in the cytoplasm, Coll2 has increased in line with the increase of LAA concentrations and the highest was revealed by LAA 100 &#956;g/mL. LAA 25, 50, and 100 &#956;g/mL resulted in higher GAG contents compared to control group (LAA 0 &#956;g/mL) (p<0.01) in the 3rd week. The increase of GAG contents in 4th week exhibited by LAA 50 &#956;g/mL (p>0.05) and LAA 100 &#956;g/mL (p<0.01). The highest calcium deposit was revealed by LAA 25 &#956;g/mL after 4 weeks of culture (p<0.01) and it was decreasing at higher concentration. This result indicated the higher concentration of LAA promoted the increase of GAG contents, whereas the lower concentration of LAA directed to hypertrophic chondrocytes indicated by the highest calcium deposit. These results indicate that LAA 100 &#956;g/mL is the most potential in chondrogenic differentiation seen from increased GAG contents and Coll2 localization.