The Choice of Anticoagulant Influences the Characteristics of Bone Marrow Aspirate Concentrate and Mesenchymal Stem Cell Bioactivity In Vitro

Abstract

Bone marrow aspirate concentrate (BMC) is commonly used as a therapeutic agent to resolve orthopedic injuries, using its unique cellularity to reduce inflammation and prime the region for repair. The aspiration of the bone marrow is performed using either sodium citrate (SC) or heparin sodium (HS) as an anticoagulant and processed via centrifugation to concentrate the cellular constituents. To date, the consideration of the impact of the two commonly used anticoagulants on the mesenchymal stem/stromal cell (MSC) population has been overlooked. The current study assesses the differences in the BMCs produced using 15% SC and HS at 1,000 U/mL or 100 U/mL final v./v. as an anticoagulant using in vitro metrics including total nucleated cell counts (TNC) and viability, the ability for mesenchymal stromal/stem cells (MSCs) to establish colony-forming units with fibroblast morphology (CFU-f), and cytokine expression profile of the MSC cultures. Our findings demonstrate that HS-derived BMC cultures result in higher CFU-f formation and CFU-f frequency at both concentrations assessed compared to SC-derived BMC cultures. In addition, there were significant differences in 27% (7 of 26) of the cytokines quantified in HS-derived BMC cultures compared to SC-derived BMC cultures with implications for MSC plasticity and self-renewal.

Figure 1

Comparison of BMC characteristics derived from 15% sodium citrate (SC) and 1,000 U/mL heparin sodium (HS) (Series 1): (a) total nucleated cell (TNC) count per milliliter, (b) nucleated cell (NC) viability, (c) colony-forming units with fibroblast morphology (CFU-f) counts per milliliter, and (d) frequency of CFU-f amongst the TNC population. Graphs display the population mean and standard error of the mean. ns: not significant, ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 2

Comparison of BMC characteristics derived from 15% sodium citrate (SC) and 100 U/mL heparin sodium (HS) (Series 2): (a) total nucleated cell (TNC) count per milliliter, (b) fold concentration of TNC count/milliliter in BMC over initial BMA, (c) nucleated cell (NC) viability, (d) colony-forming units with fibroblast morphology (CFU-f) counts per milliliter, and (e) frequency of CFU-f amongst the TNC population. Graphs display the population mean and standard error of the mean. ns: not significant, ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 3

Calcein-AM-stained CFU-fs in culture from sodium citrate- (SC-) and heparin sodium- (HS-) derived BMCs at day 5 and day 13. BMCs imaged are donor matched. Day 5 micrographs were captured at 104x total magnification, scale bar = 1,000 μm. Day 13 micrographs were captured at 52x total magnification, scale bar = 2,000 μm.

Figure 4

Comparison of BMC characteristics derived from 100 U/mL heparin sodium (100 U/mL) and 1,000 U/mL heparin sodium (1,000 U/mL). Comparisons between the respective BMCs: (a) total nucleated cell (TNC) count per mL, (b) nucleated cell (NC) viability, (c) colony-forming units with fibroblast morphology (CFU-f) counts per mL, and (d) frequency of CFU-f amongst the TNC population. Graphs display the population mean and standard error of the mean. ns: not significant, ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 5

Growth factor concentrations in cell culture media with BMCs containing 15% sodium citrate (SC) or 100 U/mL heparin sodium (HS) at day 0 (D0), day 5 (D5), and day 12 (D12): (a) angiopoeitin-2, (b) hepatocyte growth factor (HGF), (c) platelet-derived growth factor dimer AA (PDGF-AA), and (d) vascular endothelial growth factor (VEGF). Graphs display the population mean and standard error of the mean; shapes indicate donor-matched BMC products. ns: not significant, ^∗P < 0.05.

Figure 6

Immunomodulating cytokine concentrations in cell culture media with BMCs containing either sodium citrate (SC) or heparin sodium (HS) at day 0 (D0), day 5 (D5), and day 12 (D12): (a) IL-6, (b) IL-12p40, and (c) IL-23. Graphs display the population mean and standard error of the mean; shapes indicate donor-matched BMC products. ns: not significant, ^∗P < 0.05.