(L-465) Comparing the Effects of Electrical Stimulation and Electrospraying on Chondrogenesis

Introduction:: Stem cells differentiate into chondrocytes through a process called chondrogenesis. This process begins with the stem cells receiving signals from their surroundings, which causes them to change into a specific type of cell called a chondroblast. The chondroblasts then begin to produce and deposit a type of extracellular matrix called cartilage matrix, which includes collagens and proteoglycans. As the chondroblasts continue to produce and deposit cartilage matrix, they eventually become fully differentiated chondrocytes. There are several methods for differentiating stem cells into chondrocytes including growth factors, however studying physical stimulation that causes stem cell differentiation is important for several reasons: 1-Understanding the mechanisms of stem cell differentiation: Physical cues, such as mechanical forces and substrate stiffness, have been shown to play a critical role in regulating stem cell differentiation. By studying these cues, we can gain a better understanding of the underlying mechanisms that drive stem cell differentiation. 2-Enhancing stem cell therapy: Stem cell therapy holds tremendous promise for the treatment of a wide range of diseases and injuries. However, in order to fully realize the potential of stem cell therapy, we need to better understand the factors that govern stem cell differentiation. By studying physical cues, we can develop more effective strategies to promote the differentiation of stem cells into specific cell types. This work is focused on the use of alternating current (AC) on the chondrogenesis ability of human adipose-derived stem cells (hASCs).

Materials and Methods:: P4 of adipose tissue-derived stem cells (hASCs) from ScienCell (Carlsbad, California) were used for cell cultures. Cells were plated in T75 culture-treated flasks with approximately 1 million cells per flask, and culture media was changed every 3-4 days for the duration of the culture. Using NX design software and drawing from the calculations for maximum capacitance for the planned Fisher brand plates. The capacitor has two interlocking components, conscribed as the top and bottom plates, respectively, designed as a stacking cylinder. The capacitor was printed with a 3D printer. The capacitor was further modified by connecting the two steel plates to the wave generator by alligator clips directly connected to the edge of the plate. The wave generator for AC was set for 4V,12 and 20 V at 1kHZ. Cells were treated for 30 minutes once a week using a custom build capacitor that was connected to AC or DC power supply providing different voltages with a very low current.

Results, Conclusions, and Discussions:: Results and Discussion: Based on our preliminary data, we decided to treat the cells with 4v AC because it supports ECM production better than 10v AC (mRNA data). DC was not used because it gives rise to a mixed population of chondrocytes and osteoblasts. Figure 1A illustrates the electrical stimulation chamber. Cell attachment was monitored 6 hours after electrospinning to assess viability. The cells treated with Ac exhibited high cell viability, as indicated by their fluorescent green staining, with a minimal number of dead cells showing red staining. Aggrecan (Fig. 1) was observed at both D14 in treated samples but not in the control samples. Conclusion: Our group has shown previously that human adipose-derived stem cells (hASCs) that were directly incorporated into a polymer solution and electrosprayed at 10 and 15Kv, differentiated into chondrocytes without the need for any exogenous factors. The primary distinction between the present study and our previous research is that, in the current investigation, the cells are being indirectly exposed to the electrical field for a longer period. In contrast, in our previous study where the cells were electrosprayed, they were exposed to a combination of electrical field and shear stress for a short period of time[1].





Acknowledgements (Optional): : This work made use of the CytoViva enhanced, high signal-to-noise darkfield optical microscope, which has received support from the Major Research Instrumentation program (NSF MRI- 2116140) at Marshall University

References (Optional): : Nosoudi, N., et al., Differentiation of adipose-derived stem cells to chondrocytes using electrospraying. Scientific Reports, 2021. 11(1): p. 24301.