Anita Gaenko3,Dvita Bhattacharya1,Catherine Chun2,Alexander Wang4,Adam Zaidi5,Ethan Lai6,Michael Wang7,Aman Yarlagadda8,Kuan-Che Fang9,Miriam Rafailovich9
Kent Place School1,Ardsley High School2,Huron High School3,Sewickley Academy Senior School4,San Francisco University High School5,Phillips Academy6,Mounds View High School7,Newman Smith High School8,Stony Brook University, The State University of New York9
Anita Gaenko3,Dvita Bhattacharya1,Catherine Chun2,Alexander Wang4,Adam Zaidi5,Ethan Lai6,Michael Wang7,Aman Yarlagadda8,Kuan-Che Fang9,Miriam Rafailovich9
Kent Place School1,Ardsley High School2,Huron High School3,Sewickley Academy Senior School4,San Francisco University High School5,Phillips Academy6,Mounds View High School7,Newman Smith High School8,Stony Brook University, The State University of New York9
Neurodegenerative diseases such as Alzheimer’s pose a major risk to the central nervous system due to the intrinsic non-dividing nature of neurons. Therefore, it is imperative to find methods of generating functionally and morphologically neuronal-like cells from non-neuronal cells under biomimetic conditions. Previously, dental pulp stem cells (DPSCs) have been shown to differentiate into neurons via plating on Polylactic Acid (PLA), Poly(4-vinylpyridine) (P4VP), and Polylactic Acid with Resorcinol Diphosphate (RDP) Clay. A concentration of 20% RDP-Clay was found to provide the highest extent of differentiation into neurons on rough PLA surfaces [1]. Also, the usage of micro-sphere forming U-bottomed StemFit 3D suspension well plates increased the differentiability of DPSCs [2]. Therefore, in this experiment, we are investigating the effects of PLA, P4VP, and RDP Clay scaffolding on neuronal differentiation of DPSCs plated on 3D micro-sphere forming plates.<br/>DPSC micro-spheres (created using suspension well plates) were plated onto silicon wafers spin-casted with pure PLA, PLA with 20% RDP-Clay, PLA with 30% RDP Clay, pure P4VP, P4VP with 20% RDP-Clay, and P4VP with 30% RDP-Clay. These spin-coated surfaces were analyzed with Atomic Force Microscopy (AFM) to ensure homogeneous roughness across the samples so that roughness wouldn’t be a confounding factor in the growth and differentiation of the DPSCs. The RDP-Clay particles dispersed throughout the PLA with 20% RDP-Clay sample became nuclei for PLA crystals. Conversely, P4VP does not crystallize around the RDP-Clay.<br/>The plated cells were incubated for 28 days with regular media changes. EVOS images of the DPSCs were taken on Day 1, 6 days after the cells were cultured in TCP and 1 day after plating on polymer scaffolds, confirming microsphere formation following suspension well culturing.<br/>On Day 4, the elastic modulus of the DPSCs was measured for each group of polymer scaffolding via AFM. Cells plated on TCP served as a baseline for modulus. Lower modulus values indicate neuronal differentiation, as neurons are softer cells compared to DPSCs. P4VP had lower modulus (half that of TCP), which indicates that cells plated on pure P4VP had the highest elasticity and were therefore likely differentiating into neurons at a higher rate. Adding RDP-Clay concentration corresponded to a significant difference in modulus for cells plated on PV4P, but an increasing concentration of RDP-Clay was not consistent with a significant change in modulus for PLA scaffolds.<br/>On Day 28, the expression of early-stage neuronal development markers TUBB3 and NES and late-stage marker NEFM was measured using RT-PCR and compared to expression in undifferentiated DPSCs. In differentiated neuronal cells, expression of TUBB3 and NES is expected to be downregulated, and NEFM is expected to be upregulated. Corresponding to our modulus data’s implications, adding RDP-Clay to PLA did not lead to significant downregulation of TUBB3, NES, or upregulation of NEFM. However, an increasing concentration of RDP-Clay in P4VP correlated with significant downregulation of TUBB4, NES, and upregulation of NEFM in comparison to undifferentiated DPSCs, indicating that the cells likely underwent neuronal differentiation. Further data will be collected to analyze the effects of these polymer scaffoldings on the neuronal functionality of these morphologically and genetically differentiated cells.<br/>[1] Feng, K.-C., Pinkas-Sarafova, A., Ricotta, V., Cuiffo, M., Zhang, L., Guo, Y., Chang, C.-C., Halada, G. P., Simon, M., & Rafailovich, M. (2018). The influence of roughness on stem cell differentiation using 3D printed polylactic acid scaffolds. Soft Matter, 14(48), 9838-9846.<br/>[5] Bu NU, Lee HS, Lee BN, Hwang YC, Kim SY, Chang SW, Choi KK, Kim DS, Jang JH. In Vitro Characterization of Dental Pulp Stem Cells Cultured in Two Microsphere-Forming Culture Plates. J Clin Med. 2020 Jan 16;9(1):242.