CHONDROGENIC DIFFERENTIATION OF WHARTON’S JELLY MESENCHYMAL STEM CELLS (HWJ-MSCS) ON SILK SPIDROIN -FIBROIN MIX SCAFFOLD FOR CARTILAGE TISSUE ENGINEERING

• Main Author: Ratih Wahyuni, Antonia
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/46801
• Institution: Institut Teknologi Bandung

Articular cartilage tissue located in areas such as knee joints or hip joints, is devoid of vascular, lymphatic, and nervous system. The chondrocytes on the cartilage tissue are present in low density and isolated within tissue matrix. Therefore, once damage occurs to the particular area, it requires extensive treatment to restore the damage since the articular cartilage tissue has low regenerative capacity. Tissue engineering is a new alternative to repair and restore damaged cartilage tissue. There are three important factors in tissue engineering; the cells, scaffold, and bioactive compounds, which interact with each other to create new tissue. The scaffold provides structure and substrate for cells to attach, while bioactive compounds directs differentiation of the cells. In this research, the optimal concentration of bioactive coumpounds and scaffold composition were studied to support cell viability and chondrogenic differentiation. The cells were isolated from human Wharton’s Jelly (WJ) tissue, and then characterized as MSC using multipotency assay and analysis of MSC specific surface marker. Porous scaffold from mix of silk fibroin (from Bombyx morii) and silk spidroin (from Argiope sp.) were made using salt leaching method. The formed pores sturucture inside the scaffold were analysed using scanning electron microscope (SEM) images. hWJ-MSCs were grown on the silk scaffolds for 14 days to observe the biocompatibility and optimizing the silk composition by MTT assay. Bioactive compounds used were L-ascorbic acid (LAA) and platelet rich plasma (PRP). The optimal concentration of PRP and LAA to support cell viability was analysed using MTT assay result on day 1,3,5,7, and 14. The attachement of cells on the scaffold, were analyzed from immunocytochemistry (ICC) of integrin ?1 within 6, 24, and 48 hours post seeding. Integrin ?1 expression were observed using confocal microscope and the intensity was quantified with ImageJ software. hWJ-MSCs were grown on scaffold with medium containing PRP or LAA, and differentiated towards chondrogenesis for 7 and 21 days. Confirmation of chondrogenic differentiation were observed through GAG accumulation staining with Alcian Blue and production of collagen type II with confocal microscope. The isolated cells from WJ were in accorande with MSC criteria as stated by International Society for Cellular Theraphy (ICST). Scaffolds with spidroin had interconnected pore structure, except this composition SF 95% + SS 5%. Morphology of cells grown on scaffold with spidroin had already formed cell sheet and appears more elongated, while cells grown on fibroin scaffold had more rounded/spherical morphology. Optimal scaffold composition based on MTT assay result were SF 90% + SS 10%. The optimal LAA and PRP concentration in the medium were 50 ?g/ml dan 10% (v/v), respectively. Based on confocal observation of integrin ?1 expression, cells grown on scaffold with spidroin had higher integrin intensity in comparison to fibroin scaffold (P<0.05). There was an increase in integrin ?1 intensity observed between 6 hours and 48 hours after cell seeding into the scaffolds. On fibroin scaffold, the cells were forming an aggregate while on scaffold with spidroin the cells were spread evenly on scaffold surface. Confirmation of chondrogenesis by staining of GAG accumulation with Alcian Blue, showed that after 21 days of incubation, scaffold SF 90% + 10% SS with PRP 10% as induction medium had the highest GAG accumulation (P<0.05). Similar result was also observed based on the confocal images of collagen II production. These results showed that the addition of 10% silk spidroin was optimal to support cell viability, proliferation, attachment via integrin, and condrogenic differentiation of hWJ-MSCs in comparison to scaffold with 100% silk fibroin.