Human dermal spheroids combined with platelets rich plasma promote remodeling in a skin wrinkle model of aged nude mice Shiqi Hu1,2, Zhenhua Li1,2, Ke Huang2, Teng Su1,2, Ke Cheng1,2, *. 1Joint Department of Biomedical Engineering, North Carolina State University and the University of North Carolina at Chapel-Hill, Raleigh, NC; 2Comparative Medicine Institute, North Carolina State University, Raleigh, NC Statement of Purpose: Dermal (DFs) are means that they hold great potential in remodelling resident mesenchymal cells that produce and by producing more collagen. other proteins. Cultured autologous To optimize the formulation, PRP, which has attracted dermal fibroblasts are believed to reduce wrinkle attention in plastic surgeries and skin rejuvenation for formation and promote skin rejuvenation. One clinical years, was chosen as the scaffold for spheroids delivery. application for autologous dermal fibroblasts has been Figure 2 showed that PRP promoted the proliferation of approved by the Food and Drug Administration (FDA). DFs significantly in vitro. Then, in vivo study on nude Herein, three-dimensional (3D) spheroids were cultured mice was conducted. Cells injection into mice skin was to increase the therapeutic potential of DFs. In addition, performed using PBS or PRP as a carrier (Figure 3). platelets rich plasma (PRP) was used as scaffolds to Fluorescently labeled 2D DFs or 3D spheroids were enhance the viability and retention time of cells after visible for 4 days after injection. Spheroids showed a intradermal injection. This study investigated the effects much higher viability and longer retention time then 2D of self-assembled DF spheroids with the combination of DFs. What’s more, PRP groups showed an increased PRP on the anti-aging effect by reducing wrinkles on viability after injection. aged nude mice.

Figure 2. A) Representative images showing the proliferation of fibroblasts using Ki67 (red). Cells are indicated by phalloidin (green) and DAPI (blue). B) Quantification of Ki67-positive cells of control group and PRP group (n=5). All data are mean±s.d. Comparisons between two groups were performed using two-tailed unpaired Student’s t-test. *** p<0.001. Figure 1. A) Photographs of human dermal fibroblasts and the spheroids (scale bar:100 μm). B) Evaluation of vimentin (green channel) and CD90 (red channel) of 2D and 3D cultured cells. DAPI (blue) was used to locate the nuclei of the cells (scale bar:100 μm). C) Pro-collagen I expression of 2D cells and spheroid. Methods: Spheroids were derived from the self-assembly of human DFs on ultra-low attachment flasks (Figure 1A). The expression of proteins of 2D or 3D cultured cells Figure 3. Retention of 2D and 3D dermal fibroblasts on was demonstrated using immunofluorescence cell the skin of mice. 2D and 3D human dermal fibroblasts staining, ELISA and western blotting. Naturally aged were labeled with DiD and then resuspended in PBS or nude mice were used as skin wrinkle model. DF spheroids PRP for intradermal injection. In vivo imaging system were injected with PBS/PRP gel intradermally onto the (IVIS) images were taken at different time of points. neck and back skin of nude mice. Tracking of Conclusions: To summarize, the combination of PRP fluorescently (DiD) labeled cells showed the retention and dermal fibroblast spheroids was demonstrated in this time of cells, suggesting enhanced retention and study. Spheroids demonstrate elevated expression of proliferation of cells with PRP as the scaffold. mesenchymal surface markers and paracrine proteins, Results: As shown in Figure 1, spheroids showed a pro-collagen I, compared to 2D cultured cells. PRP has much-condensed structure than 2D cultured DFs. the potential to promote the proliferation of dermal Importantly, spheroids expressed higher intermediate fibroblasts and thus the remodeling of aged skin. Based filament protein vimentin (green) and mesenchymal on the in vitro and in vivo results, this study showed the surface markers CD90 (red). Specifically, to demonstrate potential use of PRP encapsulated DF spheroids in skin the anti-wrinkle ability, pro-collagen I expression level treatment. Further studies are required to verify the anti- was measured in vitro after 7 days culture. As shown in wrinkle efficiency of spheroids/PRP, such as skin scope Figure 1C, spheroids showed a three-times elevated pro- analysis before and after the treatment, histology and collagen I expression than traditional 2D cells, which western blot of skin tissues.