RESUMO
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.
RESUMO
PURPOSE: The use of bone marrow aspirate (BMA) and bone marrow aspirate concentrate (BMC) in the treatment of inflammatory orthopedic conditions has become a common practice. The therapeutic effect of BMA/BMC is thought to revolve primarily around the mesenchymal stem/stromal cell (MSC) population residing within the nucleated cell fraction. MSCs have the unique ability to respond to site of injury via the secretion of immunomodulating factors, resolving inflammation in diseased joints. Recently, the importance of hematocrit (HCT) in BMC has been debated, as the potential impact on MSC function is unknown. In the present study, we investigate MSC health over a short time-course following exposure to a range of HCT and red blood cell releasate (RBCrel) conditions. METHODS: Bone marrow-derived human MSCs in early passage were grown under conditions of 0%, 2.5%, 5%, 10%, 20% and 40% HCT and RBCrel conditions for 3 days. At each day, the percentage of viable, apoptotic and necrotic MSCs was determined via flow cytometry. Relative viable MSC counts in each condition was determined to account for dynamic changes in overall MSC densities over the time-course. Statistical analysis was performed using a one-way ANOVA comparing test conditions to the control followed by a Dunnett's multiple comparison test. RESULTS: Significant reductions in viable MSCs concurrent with an increase in necrotic MSCs in high HCT and RBCrel conditions was observed within 24 h. At each successive timepoint, the percent and relative number of viable MSCs were reduced, becoming significant in multiple HCT and RBCrel conditions by Day 3. Necrosis appears to be the initial mode of MSC death following exposure to HCT and RBCrel, followed by apoptosis in surviving MSC fractions. CONCLUSION: Various levels of HCT and RBCrel severely compromise MSC health within 3 days and HCT should be controlled in the preparation of BMC products. Further, HCT of BMCs should be routinely recorded and tracked with patient outcomes along with routine metrics (e.g. nucleated cell counts, fibroblast-colony forming units). Differences in HCT may account for the inconsistencies in the efficacy of BMC reported when treating orthopedic conditions.
