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Updated Protocols to Enhance the Regenerative Capacity of Bone Marrow

Updated Protocols to Enhance the Regenerative Capacity of Bone Marrow

E Jeffrey Donner, MD, UNITED STATES Lucanus Koldewyn, BS, UNITED STATES Sealy Hambright, PhD, UNITED STATES

Colorado Spine Institute PLLC, Johnstown, CO, UNITED STATES

2023 Congress   ePoster Presentation   2023 Congress   Not yet rated


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Summary: Important translational considerations for improving autologous biologics based on recent in vitro studies


Various factors impact the quality of autologous point-of-care biologics including their source and methods of harvest, processing and readministration to the patient. We found in vitro that: 1) the choice of anticoagulants influences the characteristics of bone marrow aspirate concentrate (BMAC) and mesenchymal stem/stromal cell (MSC) bioactivity; 2) soluble factors differ in platelets derived from separate niches of peripheral blood and bone marrow; 3) red blood cells and their releasates compromise bone marrow-derived human MSC survival; 4) MSCs in their niche have additional regenerative value.



Previous research has demonstrated the importance of the location and method of bone marrow harvest for obtaining high yields of MSCs and that contrast agents and the use of local anesthetics can decrease MSC survival.


The purpose of these studies was to improve the regenerative capacity of autologous biologic therapies. The first study evaluated the quality of PRP based on whether the source was derived from the peripheral or bone marrow blood. The second study evaluated the impact of various concentrations of two commonly used anticoagulants, sodium citrate (SC) and heparin sodium (HS), on the secretome of the MSCs. The third study investigated MSC health following exposure to a range of hematocrit (HCT) and red blood cell releasate (RBCrel). The fourth study compared the secretome of bone marrow parenchyma versus BMA fluid.


Leukocyte-poor peripheral blood-derived platelets in plasma (LPP) and leukocyte-poor bone marrow platelets in plasma (BMP) were prepared, activated, incubated and sampled at various time points. Growth and immunomodulatory factors were quantitated in LPP and BMP.

We also assessed the differences in BMCs produced using SC and HS at various concentrations using in vitro metrics including TNC and viability, the ability for MSCs to establish CFU-f, and cytokine expression profile of the MSC cultures.

Additionally, bone marrow-derived human MSCs in early passage were grown under conditions of various HCT and RBCrel concentrations. The percentage of viable, apoptotic and necrotic MSCs was determined via flow cytometry.

The bone marrow parenchyma and BMA were evaluated for MSC growth and anti-inflammatory secretome following challenges with proinflammatory cytokines.


Our findings demonstrate that platelets derived from bone marrow have a unique secretome profile compared with those derived from peripheral blood, with significant differences in anti-inflammatory cytokines, which are associated with beneficial monocyte polarization. Furthermore, various levels of HCT and RBCrel severely compromise MSC health and HCT should be controlled in the preparation of BMC products. Heparin Sulfate-derived BMC cultures result in higher CFU-f formation and CFU-f frequency at both concentrations assessed compared to Sodium Citrate-derived BMC cultures and there were significant differences in clinically relevant cytokines quantified in HS-derived BMC cultures compared to SC-derived BMC cultures with implications for MSC plasticity and self-renewal.

Most importantly, retrieving and processing bone marrow tissue with all of its constituent matrix, cellular populations including MSCs and HSCs, and other biologic components of the bone marrow niche has collectively demonstrated an improved anti-inflammatory secretome and MSC growth versus traditional BMA or BMAC.

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