(A-26) Stem cells within the Stromal Vascular Fraction Differentiate Towards an Osteogenic Lineage on Apatite-Based Materials

Introduction:: Each year, more than a half-million Americans undergo surgical reconstruction of critical-size bone defects requiring the use of bone graft materials. Autologous bone grafts, the current gold standard, require the use of a separate surgical site for autograft harvesting which is typically associated with donor site morbidity. Allogenic bone grafts can be stored for later use, but their osteoinductive efficacy is limited by the same decellularization and sterilization techniques used to prevent immunogenicity and disease transmission. Alternatively, synthetic materials that exhibit osteogenic surface properties, appropriate mechanical strength, and bio-absorbability could prove to be an ideal solution. Furthermore, current evidence indicates that supplementing these materials with stem cells could improve their regenerative properties and subsequent bone remodeling. In the current study, we investigated the effects of apatite-based, non-porous scaffolds on stromal vascular fraction (SVF) stem cell differentiation. We hypothesized that apatite-based material encourages the differentiation of stem cells within the SVF toward an osteogenic lineage.




Materials and Methods:: Several pilot studies were conducted using SVF cells harvested from fresh human adipose tissue. SVF cells were seeded on cell culture plates (control), titanium disks, and fluorapatite (FA) disks. An initial characterization was performed using FACS and immunohistochemistry prior to seeding, one week, and two weeks post seeding. In another study, harvested SVF was plated onto the same surface types and six days post collection cultured cells were harvested and pooled by their respective culture types. These cells were stained with DAPI, and live cells were obtained for single-cell RNA sequencing (scRNA-seq).

Subsequently, a cell culture study was performed to quantify the time-dependent differentiation properties of SVF cells plated on varying surfaces. SVF cells were seeded on culture plates (control), hydroxyapatite (HA) disks, and FA disks. Single-cell RNA-seq was performed for five different time points: SVF Baseline, twelve hours, four days, seven days, and fourteen days. Sequencing reads were then aligned to the reference genome using the CellRanger pipeline for each of these experiments, and Seurat was used for quality control, feature quantification, and subsequent cell clustering.




Results, Conclusions, and Discussions:: Stem cells were defined as SVF cells negative for CD45 and CD31 but positive for CD34, CD73, and CD90 using FACS analysis. Of the viable cells counted, approximately 25% of the total cells in the SVF were stem cells at baseline. By one week at least 90% of the cells on the different surfaces were stem cells, increasing to greater than 95% by two weeks. Cells on the different surfaces were stained for the osteoblast markers osteopontin (OPN) and osteocalcin (OCN). The greatest positive signals for both OPN and OCN were observed on cells that were plated onto the FA scaffolds two weeks post plating.

The scRNA-seq pilot study included 3984, 4766, and 3361 cells from control, titanium, and FA samples in the analysis. The topmost variable genes observed were LYZ, SPP1 (OPN), ACP5, MMP9, and FFABP4. Cells seeded on FA scaffolds exhibited an upregulation of calcium-binding proteins S100A4, S100A6 and S100A16, and a downregulation of multiple collagen genes compared to titanium and control samples. Compared to the control sample specifically, FA cell clusters 0 and 2 had lower cell counts, whereas clusters 3, 4, and 5 showed higher cell counts (Figure 1). Osteogenic precursor markers were upregulated in multiple clusters, including the upregulation of CRYAB in clusters 0, 1, 3, 4, 5 and 8, and the down-regulation of C1S and C1R in cluster 5. Pluripotent markers, including multiple KLF genes, were downregulated in clusters 0, 1, 2, and 5, indicating that cells in these clusters may not have committed to differentiate towards a specific cell lineage. This initial data indicates that FA may be encouraging the differentiation of SVF cells toward an osteogenic lineage. Sequencing data from the time-course study are currently being analyzed and will enable us to confirm or refute these initial observations.




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