THE ROLE OF NANOTOPOGRAPHY SUBSTRATE COATED WITH SPIDER SILK EXTRACT IN CHONDROGENIC DIFFERENTIATION OF HUMAN WHARTON'S JELLYDERIVED MESENCHYMAL STEM CELL (HWJ-MSC)

• Main Author: Khoirinaya, Candrani
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/62334
• Institution: Institut Teknologi Bandung

Cartilage is a specialized connective tissue that absorbs loads and reduces stress on synovial joints. Due to hypocellularity and avascularity, cartilage has poor regenerative capacity upon injury. Recently, mesenchymal stem cells (MSCs), has emerged as a promising option to treat cartilage defects. Human Wharton's Jelly- Derived Mesenchymal Stem Cells (hWJ-MSC) are a source of cells that are commonly used for stem cell therapy and tissue regeneration. The phenotype of hWJ-MSC is strongly influenced by its microenvironment, in particular the extracellular matrix (ECM), whose composition and structure play an important role in regulating stem cell fate. To date, the mechanisms involved in the remodeling of the ECM by hWJ-MSCs to differentiate into chondrocytes under topographical stimuli are still not clearly understood. Therefore, this study was conducted to examine the effect of nanotopography, both coated and uncoated by an extract of spider silk solution, on attachment, morphology, and growth of hWJMSC cells. The role of nanotopography and spider silk solution layer extract in hWJ-MSC chondrogenic differentiation was investigated using protein expression of chondrogenesis markers, Glycosaminoglycans (GAG), collagen type II, and SOX9, as well as mRNA expression of collagen type II and SOX9. To develop nano-engineered hWJ-MSC, polydimethylsiloxane (PDMS) was used as a substrate for printing nano-sized patterns on the surface of optical discs, Compact Disc Recordable (CD-R, 700nm) and Digital Versatile Disc Recordable (DVD-R, 350nm). Then, PDMS was plasma treated and coated with silk extract from the Argiope appensa spider. The analysis of the extract of spider silk solution includes molecular weight, viscosity, contact angle, and cytotoxicity. The extract of spider silk solution at a concentration of 50 ?g/mL proved to be non-toxic and had the best viability compared to the other groups (p<0,05). The attachment and morphology of hWJ-MSCs were analyzed using SEM and immunocytochemistry at 30 minutes and 24 hours after seeding. Meanwhile, the proliferation and differentiation of hWJ-MSCs cultured on spider silk extract-coated nanopatterns for 7, 14 and 21 days were analyzed using the MTT assay, the Alcian Blue assay, immunocytochemistry, and q-RTPCR. The attachment of hWJ-MSC to the PDMS substrate for 30 minutes showed a relatively similar morphology. The newly attached cells were still round, but after 24 hours, the filopodia began to elongate and the cells began to flatten. hWJ-MSC cultured on PDMS nanopatterns in combination with coated spider silk extract had a morphology that followed the substrate pattern and formed better cell aggregations than those grown without nanopatterns or on nanopatterns without spider silk coating. This was indicated by the growth rate (p<0,05) and an increase in glycosaminoglycans (p<0,05) compared to the non-spider silk extract-coated group. Chondrogenesis was observed through the expression of GAG, type II collagen, and SOX9. Based on the results of immunocytochemistry, hWJ-MSC underwent a change in cell shape from being elongated and aligned (day 14) to being round and forming a matrix (day 21), which was indicated by the accumulation of type II collagen and SOX 9 in the PDMS-DVD pattern. The surface of the nanopattern and the spider silk extract coating affected the attachment of hWJ-MSC to the substrate and played a role in directing the differentiation of hWJ-MSC into chondrocytes. The shape of the PDMS-DVD nano pattern is the optimal pattern to support chondrogenesis compared to the PDMS-CD nano pattern and without the pattern. Based on the results of this study, the PDMS-DVD nanopattern coated with spider silk extract solution has the potential as a new method for tissue engineering tissue by directing the chondrogenic differentiation of hWJ-MSC.