Spicules:
Fragments, also known as spicules, are aggregates of bone marrow cells that are pulled from the bony matrix of the marrow space (trabecular) during the aspiration process. They represent what will be seen on the bone marrow biopsy as material that came from what was in between the bony trabecular. Many of these aggregates are smaller than 200 micron filter so, you are not filtering many of them out anyway but you do lose them with a centrifugation protocol. Typical filter is capturing fat and clots. Bone Chunks would not fit through the 22 gauge needle or the side ports of the aspiration needle. These cell aggregates are VERY rich in MSC and HSC. (1,2)
1) Ahmadeigi et al ;The aggregate nature of human mesenchymal stromal cells in native bone marrow. Cytotherapy 2012 Sep;14(8):917-24.
2) Ahmadbeigi et al Isolation, Characterization, and Transplantation of Bone Marrow Derived Cell Components with Hemtopoietic Stem Cell Niche Properties Stem Cells Dev. 2013 Dec 1; 22(23): 3052–3061.
RBC Count:
The hematocrit of the product from the needle will be the native hematocrit and will be equal to that of centrifugation protocols but will have a slightly higher number of nucleated red blood cells. Red cells are a natural part of any clot and play a role in the regenerative process; it is not necessary to remove them.
- Red cell are nitric oxide sinks ( ref 1)
- Nitric Oxide is a potent stem cell stimulant (ref 2)
- Red Cells release iron
- Iron is used by cells under stress to survive and function (ref 3)
- Stem cells reside deep in the red cell layer including very small embryonic-like cells are discarded during centrifugation . (ref 4, 5, 6)
1) Cortese et al Endothelial nitric oxide synthase in red blood cells: Key to a new erythrocrine function Volume 2, 2014 251-258 http://www.sciencedirect.com/science/article/pii/S221323171400010X
2) Alexandra Aicher Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells Nature Medicine 9, 1370 - 1376 (2003) Published online: 12 October 2003;
3) Gardner, et al Superoxide Radical and Iron Modulate Aconitase Activity in Mammalian Cells June 2, 1995 The Journal of Biological Chemistry, 270
4) Ahmadbeigi et al The aggregate nature of human mesenchymal stromal cells in native bone marrow. Cytotherpy 2012 Sep;14(8):917-24.
5) Juopperi et al Isolation of Bone Marrow Derived Stem Cells using Density-Gradient Separation Experimental Hematology 35 (2007) 335-341
6) Bhartiva D et al “Very Small Embryonic-Like Stem Cells with Maximum Regenerative Potential Get Discarded During Cord Blood Banking and Bone Marrow Processing for Autologous Stem Cell Therapy” Stem Cells and Development Volume 221 NO 1 2012
In Vivo Data:
Lastly, this established science corresponds to published in-vivo clinical experience where marrow of a native hematocrit was used to treat knee cartilage defects. No adverse reactions were noted when a native level of marrow cells was used. (1,2)
1) Gobbi A. Et al “One-Step Cartilage Repair with Bone Marrow Aspirate Concentrated Cells and Collagen Matrix in Full-Thickness Knee Cartilage Lesions; Results at 2-Year Follow-up” Cartilage. 2011 Jul; 2(3): 286–299A.
2) Fortier L. et al. “Concentrated Bone Marrow Aspirate Improves Full-Thickness Cartilage Repair Compared with Microfracture in the Equine Model” J Bone Joint Surg Am. 2010 Aug 18;92(10):1927-37
WBC Count:
When an aspirate is performed poorly ( i.e. single stick draw using a vac-syringe, aspirate all from one location) the aspirate is comprised mostly of peripheral blood. The density of the float in the marrow kits is greater than the PRP kits and so the BMAC kit will capture more red cells and granulocytes. In the case of a poor aspirate, the only difference between PRP and BMAC, since they are both being made from peripheral blood, is that the BMAC has higher red cells and granulocytes. With a poor aspirate, the higher nucleated cell counts in the centrifugation protocols is due to higher cells from the peripheral blood and not marrow cells. The published literature looks at the number of CFU-F in the marrow product, whether centrifuged or not, to determine the quality of the biologic because CFU-F is found in marrow but not peripheral blood. Clinical results in these peer reviewed journal articles for different muscular skeletal dis-orders were lined to CFU-F in the graft, not the nucleated cell content.
Centrifugation / Concentration:
Many cell aggregates from the marrow aspirate are as dense as a mature red cell. (1,2) This is the reason why centrifugation protocols get such a low percent recovery of mesenchymal stem cells (CFU-F); they are thrown away with the red cells. Quote from ref 3 below: “The isolated cells from both the upper and lower fractions were characteristic of MSC. Although it is commonly believed that MSC are single suspending mononuclear cells and so are enriched in the upper fraction of Ficoll-Paque after density-gradient separation, our data showed that considerable numbers of these cells were accumulated in the lower fraction.” (3) Lastly, whole blood is used for epidural patches; if red cells were inflammatory, they could not be used near such sensitive tissues.
1) Juopperi TA, Schuler W, Yuan X, Collector MI, Dang CV, Sharkis SJ. Isolation of bone marrow-derived stem cells using density-gradient separation. Experimental hematology 2007;35:335-41.
2) Bhartiya D, Shaikh A, Nagvenkar P, et al. Very small embryonic-like stem cells with maximum regenerative potential get discarded during cord blood banking and bone marrow processing for autologous stem cell therapy. Stem cells and development 2012;21:1-6.
3) Bhartiva D et al “Very Small Embryonic-Like Stem Cells with Maximum Regenerative Potential Get Discarded During Cord Blood Banking and Bone Marrow Processing for Autologous Stem Cell Therapy” Stem Cells and Development Volume 221 NO 1 2012