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Selective Surface-Induced Osteogenic Differentiation Of Stromal Vascular Fraction On Fluorapatite Scaffolds
Andrew Miller, PhD, Jill Shea, PhD, Peter Beck, MD, Jay Agarwal, MD, Sujee Jeyapalina, PhD.
University of Utah, Salt Lake City, UT, USA.

PURPOSE:Critical-size bone defects are defined as defects that are unlikely to heal spontaneously despite stabilization of the site. These defects often require surgical intervention and bone grafts. Autologous bone grafts serve as the gold standard for these types of interventions but require a separate surgical site for harvesting autograft, and, such autologous material is limited in supply. Synthetic bone grafts, such as ceramic scaffolds, could be an ideal alternative if they exhibit osteogenic surface properties, desired mechanical strengths, and serializability. In addition, stromal vascular fraction (SVF) could be used to provide the necessary source of stem cells for improving the remodeling potential of such scaffolds. In this study, we use single-cell RNA-sequencing (scRNA-seq) to assess the osteogenic potential of fluorapatite (FA) and hypothesized that FA scaffolds encourage the osteogenic differentiation of stem cells within SVF METHODS:
Fresh human adipose tissue was obtained using the institutionally approved IRB (IRB#1094). The SVF was isolated using collagenase I and II and Lysis buffer to lyse the red blood cells prior to centrifugation. Isolated SVF was seeded on 8 wells of a cell culture plate (control), 12 titanium disks, and 20 fluorapatite (FA) nonporous scaffolds controlling for surface area by plating the same density of cells on each surface (80,000 cells/1.9cm2). After culturing for 6 days, samples were pooled by culture type, and scRNA-seq was performed. CellRanger and Seurat were used for sequence alignment, quality control, feature quantification, and clustering, while SingleR was used for cell-type annotation based on the cell transcription profiles.RESULTS:
After quality control, 3397, 4435, 2983 cells for control, titanium, and FA samples were included for further analysis. The topmost variable genes observed in our dataset were LYZ, SPP1 (OPN), ACP5, MMP9, and FFABP4. FA cells exhibited upregulation of calcium-binding proteins S100A4, S100A6, S100A16 and downregulation of multiple collagen genes compared to titanium and control samples. Clustering and cell-type annotations are shown in Figure 1. Clusters 0 and 2 exhibited the most significant decrease in cells, whereas clusters 3, 4, and 5 showed the greatest increase on FA surfaces. Additionally, osteogenic precursor markers were expressed in multiple clusters, including upregulation of CRYAB in clusters 0, 1, 3, 4, 5, 8, down-regulation of C1S and C1R in cluster 5, and downregulation of TGFB1 in clusters 1, 3, 4, 5, and 6. Additionally, pluripotent markers, including multiple KLF genes, were downregulated in clusters 0, 1, 2, 5. CONCLUSION:
In the current study, individual transcriptomes of SVF cells plated on control, titanium, and FA samples were obtained using scRNA-seq. Results highlighted the ability to identify cell-type clusters, including osteogenic precursor markers. Additionally, osteogenic precursor markers were identified in multiple clusters indicating that a time-course study may be necessary to further understand the kinetics of osteogenic differentiation of the SVF. Regardless, our data seemed to suggest that stem cells are undoubtedly directed to the osteogenic lineage.


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