COMBINATION OF BLURAY-DISC RECORDABLE (BD-R) OPTICAL DISC BASED NANOPATTERN AND SPIDROIN FOR APPLICATIONS IN CARTILAGE TISSUE ENGINEERING FROM HUMAN WHARTON'S JELLY-DERIVED MESENCHYMAL STEM CELLS

• Main Author: Hendra Punja Unggara, Acep
• Format: Theses
• Language: Indonesia
• Subjects: Teknologi
• Online Access: https://digilib.itb.ac.id/gdl/view/84377
• Institution: Institut Teknologi Bandung

Nanopatterning is a technique for creating nanopatterns or nanometer scale substrate engineering on the surface of materials which is widely developed for tissue engineering applications. This development was carried out because the cell microenvironment in the body has nanoscale features in the form of nanotopography. Nanotopographic engineering aims to mimic the in vivo environment of cells by utilizing the principle of cell interaction with nanopatterns on the surface of the substrate. The interactions that occur produce responses in the form of geometric cues and mechanical forces that influence cell development, including the processes of adhesion, migration, proliferation and differentiation. Both responses are transmitted as mechanical signals from the substrate to the nucleus, becoming a molecular process collectively known as mechanotransduction. Human Wharton's Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) is a cell source that is widely used in tissue engineering studies by directing their multipotent ability to differentiate into several cell types. The phenotypic expression of hWJ-MSCs cells is greatly influenced by the modulation of biophysical factors in their microenvironment. Several studies have proven that engineered nanotopography's stimulus effect as a substrate can induce the differentiation process of hMSCs. The research aims to test the effect of nanopattern-based nanotopography coated or not coated with spidroin extract from A. appensa spider webs on the attachment, spreading, morphology, growth and differentiation of hWJ-MSCs cells into chondrocyte cells. The research began by carrying out the primary culture of hWJ-MSCs cells obtained from donors from caesarean section patients. The primary culture of passage four hWJ-MSCs cells were then analyzed for positive and negative markers using flow cytometry, and their multipotency capabilities were under the International Society of Cellular Therapy (ISCT) requirements. To develop nanopattern-based nanotopography, Polydimethylsiloxane (PDMS) polymer was used as a substrate to print nano patterns on the surface of Bluray optical discs (BR-D) polycarbonate which is sized for 130-230 nm. The PDMS-BD-R nanopattern substrate was treated plasma and coated with spidroin silk extract. The characterization of the nanopatterns formed was analyzed using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Spidroin was characterized by determining several parameters such as viscosity, zeta potential, raman spectroscopy, contact angle and cytotoxicity test (MTT assay). The interaction of hWJ-MSCs cells with nanopatterns was analyzed using SEM in the first 48 hours after cell seeding. The proliferation of hWJ-MSCs cells cultured on PDMS-BD-R nanopatterns and controls, coated and uncoated with spidroin extract, was analyzed using the MTT assay. Meanwhile, the chondrogenesis process was analyzed by determining the expression of specific markers, such as the abundance of matrix Glycosaminoglycan (GAG), using Alcian Blue staining and Immunocytochemistry of Type II Collagen and SOX9 on days 7, 14 and 21. Based on the research results, primary culture passage 4 of hWJ- cells MSCs have fulfilled the ISCT requirements with fibroblast-like morphology and plastic-adherent propertie. The multipotency tests show the cells can differentiate into adipocytes, chondrocytes and osteocytes and have positive markers CD90 (96.6%), CD73 (99%), CD105 (91.2%); Linnnegative markers (0%) for CD34, CD45, CD11b, CD19. The characteristics of the spidroin extract solution have viscosity and zeta potential values of 0.896 mPa.s and -34.8 mV, respectively. In contrast, the cytotoxicity test at spidroin concentrations of 50 and 100 ?g/mL shows the hWJ-MSCs cell proliferation rate is >125%, which indicates spidroin is not toxic to cells. The test results of the spidroin contact angle on the PDMS-BD-R+Spidroin substrate are 14.5620, and the results of Raman Spectroscopy analysis shows that there is a possible distribution of RGD sequences in the wave number range of 900-1100 cm-1 and 1250-1525 cm- 1. The PDMS-BD-R nanopattern fabrication has a nanogroove width of 234 ± 8.92 nm, a nanoridge width of 145 ± 2.67 nm and a peak height of 15 ± 0.782 nm. The process of attachment and spreading, as well as the morphology of hWJ-MSCs cells cultured on the PDMS-BR-D+Spidroin substrate for 48 hours, shows a more significant number of cells extending their filopodia and lamellipodia; the cells grow following a pattern, and the cell density is higher when compared with a group of cells cultured on non-pattern PDMS+Spidroin. This is in line with the MTT proliferation results that there is an increase in hWJ-MSCs cell proliferation in the group of cells cultured on the PDMS-BD-R+Spidroin substrate until day 14 with p-value < 0.001 compared with the control group. Matrix abundance test (Alcian Blue Staining) shows that the cell group cultured on PDMS-BDR+ Spidroin experienced an increase in GAG matrix abundance (p<0.001) when compared to the control group on days 14 and 21. Based on the results of Immunocytochemistry, Collagen type II and SOX9 are detected on day 7, then their fluorescence intensity increased (days 14 and 21) and hWJ-MSCs cells experienced a change in cell shape from previously elongated and lined (day 7) to round and shaped chondrocyte nodule aggregates (days 14 and 21) in groups of cells cultured on PDMS-BD-R+Spidroin substrate. Based on the research results, it can be concluded that the combination of nano topography and spidroin extract coating can increase the attachment, spreading and proliferation of hWJ-MSCs cells, accelerate the formation of chondrocyte nodule aggregates and facilitate the differentiation process of hWJMSCs cells into chondrocyte cells as a new method in cartilage tissue engineering.