At the 2018 Interventional Orthopaedic Foundation meeting which is a multidisciplinary collection of clinicians interested in the advanced field of interventional orthopaedics and in the science and application of orthobiologics,  a faculty assignment was to discuss the many reasons why centrifuging and concentrating bone marrow aspirate makes good clinical sense when compared to just using bone marrow aspirate alone as a treatment.  Below is a summary of the talking points..

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7 Reasons to Centrifuge and Concentrate Bone Marrow Aspirate

Why would a physician use BMA/BMC over PRP for regenerative treatments?

Bone marrow aspirate (BMA) contains MSCs (Stem Cells)  (blood has none) and significantly elevated levels of HSCs (Hematopoietic stem cells) and IRAP (Interleukin-1 Receptor Antagonist Protein) compared to blood.

BMA still has platelets and plasma proteins like blood.

 1.  Can improved bone marrow aspiration technique compensate for not centrifuging the BMA?

No.  Hernigou et al found that MSCs or CFU-Fs (colony forming units-fibroblasts) could be enriched more than 3x by using small volume syringes with rapid generation of negative pressure. This is great for harvesting a better quality BMA, but stopping short of centrifugation to save time or money is short sighted because it ignores:

Further enrichment of MSCs above however rich the BMA is can be achieved by centrifugation and isolation of the buffy coat.

Many systems can get you get 4-10x over baseline BMA values for all WBCs, including MSCs.

If maximizing MSCs is the goal, why not maximize both the aspiration and concentration?

2.  Does small volume aspiration increase other cells or platelets?

No!   There is no data suggesting that better aspiration increases other types of cells in the BMA.

HSCs and Platelets are only enriched by centrifuging a larger volume down to a smaller volume. If platelets are not increased, neither are platelet-bound cytokines (PDGF, VEGF, etc.).

The contribution of HSCs to MSK regenerative medicine is not as understood as MSCs, but it is widely believed that HSCs play a synergistic role in immunomodulation.

3. Does bone marrow aspirate get rid of clots, debris, or fat from the marrow?

No!   Exclusion of clots, bone debris, fat, and other pieces of tissue that would be filtered out prior to centrifugation or would separate from the buffy coat during centrifugation.

Using raw BMA means injecting these elements into inflamed joints or discs in a certain number of patients.

4.  Can you get rid of the red blood cells in the end product if you don’t concentrate?

No!   You cannot reduce hematocrit without centrifuging and selecting out the buffy coat layer.

There is no consensus on the role of RBCs as inflammatory or anti-inflammatory, but generally RBCs are excluded from the injection volume in favor of more buffy coat and plasma.

If a practitioner felt RBCs in BMA posed no issue, why not inject whole blood rather than PRP in soft tissue injuries?

5. Which regenerative elements can be concentrated in BMC above baseline BMA via centrifugation?

• MSCs
• HSCs
• Platelets
• Indirectly, the “dose” of Platelet-derived cytokines (VEGF, PDGF, FGF, etc.) in the product maybe elevated because there is a greater concentration of platelets (>4x)

6. Which elements in BMA or blood are not enriched by centrifugation?

PLASMA PROTEINS!

• IRAP (secreted by activated monocytes, macrophages, and neutrophils)
• A2M (synthesized primarily in the liver)
• Fibrinogen (synthesized primarily in the liver)

The average protein’s mass is less than one billionth of a mammalian cell, so centrifugation in the clinical setting (<2500 G’s) does not cause a separation or settling of proteins from the plasma. Ultracentrifugation at >100,000 G’s would be required to concentrate IRAP, A2M, and other plasma proteins by spinning alone.

7. Can you enrich plasma proteins at point of care in bone marrow aspirate?

No!   Removing water from plasma results in higher amounts of proteins (fibrinogen, A2M, IRAP) per volume. Plasma is obtained after centrifugation.  There is mounting published evidence that plasma proteins like A2M and IRAP play an important role in the clinical success of our therapies.

Trying to ultrafilter uncentrifuged blood or BMA is not feasible due to the viscosity of cells in the fluid. Freeze-drying whole blood or BMA would lyse and kill cells and leave intracellular debris that may be inflammatory or apoptotic. Ultracentrifugation and lyophilization are not feasible in a clinical setting anyway.

IN CONCLUSION

• The only advantage to not centrifuging is saving time (<15 minutes) and possibly kit costs depending on which needle or kit you use.
• Great aspiration technique (ie. small syringes and small volume draws, or the Marrow Cellutions needle) may enrich MSCs by 3x, BUT centrifugation can easily achieve another 4x on top of that or 12x above the poor aspiration technique baseline BMA.
• HSCs and platelets are only enriched, while RBCs, clots, and bone fragments are only excluded, by centrifugation.
• Going beyond aspiration and centrifugation, filtration is the only way to further enrich plasma proteins like IRAP, A2M, and fibrinogen. Centrifugation is required to obtain the platelet-poor plasma to enrich plasma proteins in the end product.
• At the Dallas PRP and Stem Cell Institute they have had no infections in hundreds of bone marrow aspirations.
• At the Dallas PRP and Stem Cell Institute it takes approximately 1 hour start to finish to have the procedure…including the 10 minute centrifuge time!
• Published literature with human patients where bone marrow was used as a source of stem cells is completely dominated by studies that use bone marrow concentrate and not just bone marrow aspirate.
• Ultimately, with peer reviewed evidence, there may be a place for more orthobiologics that are useful in patient care, but when it comes to bone marrow aspirate, for all of the reasons listed above…..The choice is to concentrate the bone marrow every time.
  • Get a higher CFU (colony forming unit) count (3x-5x better than a great bone marrow aspiration that doesn’t concentrate)
  • Concentrate the plasma proteins
  • Able to remove the red blood cells
  • Remove any debris
  • It takes 1 hour in the office under ultrasound guidance
  • There have been no infections and no complications other than occasional soreness more than a day at the donor site from our practice tested single stick technique.