*6.1.2 Two-step BMA centrifugation protocol*

It is our belief that the preparation of a vial of concentrated MSCs is best created by the so-called double-spin protocols, using dedicated and approved disposable concentration devices. BMA centrifugal processing techniques, to produce a viable BM-MSC injectate, are generally accepted methods when executed at POC, because these preparation protocols seek to overcome the limitations of MSC ex vivo cell culturing techniques. In this section we touch on a BMC preparation protocol to produce PurePRP SupraPhysiologic Bone Marrow Concentrate (PureBMC®SP, EmCyte Corporation, Fort Myers, FL, USA). The PureBMC®SP autologous biologic is part of an autologous cellular platform technology, facilitating the preparation of platelet-rich plasma and adipose tissue concentrates. A two-step centrifugation and preparation protocol will concentrate the indispensable BMA cellular content to a BMC. Following a first centrifugation spin, the BMA is sequestered in a BM plasma fraction (BMPF), containing a buffy coat layer and RBCs. The BMPF is

**23**

*The Rationale of Autologously Prepared Bone Marrow Aspirate Concentrate for use…*

volume varying between 4- and 10-fold the native concentrations.

**6.2 Cellular differences between BMA and BMC injectates**

are significantly decreased when compared to a BMA injectate.

encumbering clinical outcomes, is presented in **Figure 12**.

*6.2.2 Comparative laboratory data BMA vs. BMC*

*6.2.1 Red blood cells and hemolysis*

aspirated, immediately followed by a separate collection of 2 ml of RBCs, following the instructions for use of the PureBMC® concentration device. Both volumes are then transferred for a second centrifugal spin cycle to the concentration compartment of the same device. During the second spin, a specific centrifugation protocol is accomplished, leaving the bone marrow cells in a concentrated fashion attached at the bottom of the chamber. Excessive BMPF is manually removed, leaving behind a specific BMC volume for resuspension. The amount of this volume depends on the requirements for clinical applications. Therefore, the BMC injectate volume may vary between 3 and 10 ml, with increased cell concentrations according to this final

In a BMA injectate, the concentrations of the cells resemble the concentration of the cells that are present in the bone marrow cavity. However, based on aspiration techniques, the number of MSC might be increased. A BMC is a small volume of fluid containing a high concentration of cells extracted from the bone marrow, such as high yields of MSCs (can be measured as CFU-Fs), HSCs, progenitor cells, total nucleated cells, and platelets, at a significant concentration above BMA baseline values. Furthermore, the heterogenous nature of marrow cells is completed by the presence of increased levels of growth factors [106, 107], cytokines like IL-8, and interleukin-1RA [94]. Additionally, in a BMC injectate following a two-step centrifugation procedure, the RBC and plasma-free hemoglobin (PFH) concentrations

Throughout the aspiration procedure, RBCs can be damaged as a result of high shear forces [108]. As a consequence, the RBC membrane will start to disintegrate, and hemolysis, with the release of PFH, will occur. Damaged RBCs and free hemoglobin (Hb) lead to the development and release of toxic Hb forms, like free hemin, ferric Hb, and iron [109]. This is of particular concern as PFH and their split products, heme and iron, cannot be cleared, by natural scavenger proteins, when bone marrow injectates are applied in any microenvironment, as these are outside of the blood stream. A graphic representation of the pathophysiological effects and reactions of PFH, leading to various hemolytic-related sequelae and potentially

In **Table 1** the effects of concentrating BMA to BMC with regard to some of the most important marrow constituents and factors are shown, as discussed in Section 4.1. The data in the table represent a clinical bilateral BMA model, using two different harvesting systems. For both systems, BMA was aspirated in an identical manner, at three different depth levels collecting in total 10 ml of marrow. Furthermore, to compare the cellular differences between a BMC injectate and a BMA injectate (BMA-MC), we collected an additional 40 ml of BMA with the Aspire system, after the first 10 ml. This allowed for a total processing volume of 60 ml to produce BMC. Laboratory analysis resolved that both BMA devices were almost similar with regard to cell viability and numbers. Interestingly, with regard to CFU-Fs, the data are in accordance with Hernigou [40], and the first marrow aspiration provides the highest number of CFU-Fs. However, when comparing the BMA-MC cellular composition (a patient treatment specimen) with the BMC treatment specimen,

*DOI: http://dx.doi.org/10.5772/intechopen.91310*

*The Rationale of Autologously Prepared Bone Marrow Aspirate Concentrate for use… DOI: http://dx.doi.org/10.5772/intechopen.91310*

aspirated, immediately followed by a separate collection of 2 ml of RBCs, following the instructions for use of the PureBMC® concentration device. Both volumes are then transferred for a second centrifugal spin cycle to the concentration compartment of the same device. During the second spin, a specific centrifugation protocol is accomplished, leaving the bone marrow cells in a concentrated fashion attached at the bottom of the chamber. Excessive BMPF is manually removed, leaving behind a specific BMC volume for resuspension. The amount of this volume depends on the requirements for clinical applications. Therefore, the BMC injectate volume may vary between 3 and 10 ml, with increased cell concentrations according to this final volume varying between 4- and 10-fold the native concentrations.

#### **6.2 Cellular differences between BMA and BMC injectates**

In a BMA injectate, the concentrations of the cells resemble the concentration of the cells that are present in the bone marrow cavity. However, based on aspiration techniques, the number of MSC might be increased. A BMC is a small volume of fluid containing a high concentration of cells extracted from the bone marrow, such as high yields of MSCs (can be measured as CFU-Fs), HSCs, progenitor cells, total nucleated cells, and platelets, at a significant concentration above BMA baseline values. Furthermore, the heterogenous nature of marrow cells is completed by the presence of increased levels of growth factors [106, 107], cytokines like IL-8, and interleukin-1RA [94]. Additionally, in a BMC injectate following a two-step centrifugation procedure, the RBC and plasma-free hemoglobin (PFH) concentrations are significantly decreased when compared to a BMA injectate.

#### *6.2.1 Red blood cells and hemolysis*

*Regenerative Medicine*

MSC homing [66].

it is critically important for MSCs to control cell adhesion in the ECM of the treated tissue. This will occur through the expression of fibronectin and specific integrin and selectin protein adhesion molecules, which are binding to collagen and fibrin ECM components [102], initiating tissue healing and regeneration through cell adhesion, cell growth, migration, and differentiation [104]. The migration ability of MSCs is further controlled by a wide range of growth factors, acting under the receptor tyrosine kinase signaling principle [105], once more illustrating the importance and presence of platelets and their growth factors in the collected BM vial. Furthermore, the administration of MSCs via various delivery routes (intravenous, intraperitoneal, intra-arterial, in situ injections) seems to have an effect on

When applying regenerative medicine MSC applications, physicians have a choice to use either a BMA as a regenerative injectate, without any processing steps, or they can harvest a particular BMA volume necessary to produce a BMC, with dedicated devices and centrifuges. Additionally, the differences between a BMA

In the freshly aspirated BMA samples, the heterogenous cellular content is

Prior to a BMA procedure, it is recommended that bone marrow harvesting devices, concentration devices, and all of the processing accessories that will be in contact with BM are subject to a thorough heparin rinsing. Furthermore, several instructions for use advice to leave a volume of anticoagulant in the aspiration syringes and processing device as well, as BM tissue has the potential for rapid clotting. Before a BMC concentration device is loaded for processing, the aspiration syringes volumes are transferred into one consolidating collection syringe and subsequently filtered through a 200u heparin rinsed filter to eliminate particles,

It is our belief that the preparation of a vial of concentrated MSCs is best created by the so-called double-spin protocols, using dedicated and approved disposable concentration devices. BMA centrifugal processing techniques, to produce a viable BM-MSC injectate, are generally accepted methods when executed at POC, because these preparation protocols seek to overcome the limitations of MSC ex vivo cell culturing techniques. In this section we touch on a BMC preparation protocol to produce PurePRP SupraPhysiologic Bone Marrow Concentrate (PureBMC®SP, EmCyte Corporation, Fort Myers, FL, USA). The PureBMC®SP autologous biologic is part of an autologous cellular platform technology, facilitating the preparation of platelet-rich plasma and adipose tissue concentrates. A two-step centrifugation and preparation protocol will concentrate the indispensable BMA cellular content to a BMC. Following a first centrifugation spin, the BMA is sequestered in a BM plasma fraction (BMPF), containing a buffy coat layer and RBCs. The BMPF is

pervasively distributed in the vial, as long as clotting is prevented.

**6. Bone marrow aspirate aspiration and processing**

injectate only and a BMA concentrate are discussed.

**6.1 BMA-MSC procedural steps**

*6.1.1 Anticoagulation protocol*

fibrin strands, and fat tissue.

*6.1.2 Two-step BMA centrifugation protocol*

**22**

Throughout the aspiration procedure, RBCs can be damaged as a result of high shear forces [108]. As a consequence, the RBC membrane will start to disintegrate, and hemolysis, with the release of PFH, will occur. Damaged RBCs and free hemoglobin (Hb) lead to the development and release of toxic Hb forms, like free hemin, ferric Hb, and iron [109]. This is of particular concern as PFH and their split products, heme and iron, cannot be cleared, by natural scavenger proteins, when bone marrow injectates are applied in any microenvironment, as these are outside of the blood stream. A graphic representation of the pathophysiological effects and reactions of PFH, leading to various hemolytic-related sequelae and potentially encumbering clinical outcomes, is presented in **Figure 12**.

#### *6.2.2 Comparative laboratory data BMA vs. BMC*

In **Table 1** the effects of concentrating BMA to BMC with regard to some of the most important marrow constituents and factors are shown, as discussed in Section 4.1. The data in the table represent a clinical bilateral BMA model, using two different harvesting systems. For both systems, BMA was aspirated in an identical manner, at three different depth levels collecting in total 10 ml of marrow. Furthermore, to compare the cellular differences between a BMC injectate and a BMA injectate (BMA-MC), we collected an additional 40 ml of BMA with the Aspire system, after the first 10 ml. This allowed for a total processing volume of 60 ml to produce BMC. Laboratory analysis resolved that both BMA devices were almost similar with regard to cell viability and numbers. Interestingly, with regard to CFU-Fs, the data are in accordance with Hernigou [40], and the first marrow aspiration provides the highest number of CFU-Fs. However, when comparing the BMA-MC cellular composition (a patient treatment specimen) with the BMC treatment specimen,

#### **Figure 12.**

*Pathophysiological effects and reactions of RBCs in BMC vials. In absence of scavengers and compensatory mechanisms, PFH split products can lead to toxic consequences like inflammation and prooxidant effects, endothelial cell dysfunction, and vasoconstriction. Biological treatment specimen, containing high concentrations of RBCs, will lead to RBC cell membrane disruption (eryptosis,) releasing macrophage migration inhibitory factor (MIF) (courtesy of P. Everts and modified from Schaer et al. [109]).*

significant differences occur. Centrifugation foremost significantly increases cells who take part in regenerative processes when compared to the BMA-MC product. In contrast the non-regenerative RBCs and PFH concentrations are significantly reduced in the treatment vial, while maintaining a higher cell viability after centrifugation. Our findings, with regard to cellular enrichments comparing a BMA with a BMC, are in agreement with others [41, 110], but not regarding RBC content and PFH. The cell concentrations are not only depending on the centrifugation protocols and the final BMC volume but are contingent of a meticulously executed BMA procedure, maintaining high cell viability, with minimal cell destruction.
