**4. Bone marrow aspiration procedures**

*Regenerative Medicine*

adipocytes [34].

expansion [35].

**3.4 Extracellular matrix**

*3.3.3 Megakaryocyte niche*

*3.3.2 Perivascular niche*

The BM is highly vascularized, with large central arteries branching into progressively smaller microvessels like arterioles and transitioning into venous sinusoids near the bone (endosteal) surface. Therefore, it has been suggested that HSCs are maintained in a perivascular niche by endothelial or perivascular cells, as they are frequently located adjacent to the blood vessels [29]. These occurrences resulted in the expression of various perivascular mesenchymal cell makers CD146, stromal cell-derived factor-1 (SDF-1) also referred to as CXCL12, and Nestin-GFP, defining the heterogenous BM stroma cell composition [9], including the MSCs that surround the blood vessels [30]. The more perivascular nature of MSC niches was validated by Shi and Gronthos, demonstrating the expression of α-smooth muscle actin (αSMA) at perivascular sites, with the immunohistochemical localization of specific CD marker cells [31]. Mores studies confirmed the presence of MSCs in BM, expressing a Nestin-GFP transgene, localized and attached around the BM blood vessels and part of the perivascular HSC niche [32]. Kunisaki et al. indicated that most HSCs do not only have a perivascular presence, but they are preferentially located in the BM endosteal regions. The endosteal regions contain a complex network of stromal cells as well that have been implicated in HSC maintenance, including arteriolar and venous endothelial cells, pericytes, and chemokine (C-X-C) ligand 12 (CXCL12) reticular cells. Their study also suggested that quiescent HSCs localize preferentially to small arterioles near the endosteum, suggesting that distinct niches may exist for both quiescent and proliferating HSCs [33]. From all these findings, it can be concluded that pericytes are in fact MSCs, because they can differentiate in osteoblasts, chondrocytes, and

Megakaryocytes (MK) are the precursor cells of blood platelets. BM hematopoietic cells are responsible for platelet production. MK may regulate HSCs indirectly as they are closely associated with BM sinusoidal endothelium, extending cytoplasmic protrusions into the sinusoids to produce platelets. A direct regulation of HSC by MK through signaling of transforming growth factor beta 1 was established, with activation of quiescent HSCs and increased proliferation rate. In the event of a sudden response to systemic stress signaling, fibroblast growth factor-1 as part of the MK growth factor pool will start signaling HSCs and will overshadow the TGF-b1 signaling in order to stimulate high volumes of HSC

The role and function of the extracellular matrix (ECM) can be defined as key structural-functional components of cell niches, including soluble factors, cell-cell contacts, and cell-matrix adhesions present in these microenvironments. ECM components include fibrillar proteins, with, among others, collagen fibers, fibronectin, and other filamentous network components. The ECM's mechanical stability is provided by collagen [36]. Other significant ECM components supporting the BM niches are glycosaminoglycans and mainly hyaluronic acid via its receptor CD44. The surface marker is also expressed by MSCs and HSCs [37]. Intracellular signaling in the ECM occurs through cytokine and growth factor membrane receptors, similar to the MSC niche. These cytokines and receptor activities contribute to cross

**6**

Exploiting BM preparations at POC seeks to overcome the limitations of ex vivo MSC culturing. Clinicians utilizing regenerative medicine applications have a growing interest in using the concentrated bone marrow products, since it is well acknowledged that BM is a plentiful source of MSCs, progenitors, and other cells residing in the trabecular part of flat and long bones, acquired via appropriately performed BMA procedures [40, 41]. The regenerative medicine market is rapidly growing, with many procedures performed in musculoskeletal, orthopedic, and spinal disorders, wound care management including critical limb ischemia, and tissue engineering [42–45]. Several groups have mentioned some considerations when performing BM harvesting procedures, addressing a variety of factors that have an impact on patient comfort and the quality of the harvested BM. Emphasis was given to procedural safety issues when using harvesting needle systems, level of experience of the operator, the choice for concentration technology and centrifugation devices, and pain management [46]. Autologous regenerative medicine BM-MSC applications may range from a harvesting a low volume of BM and direct, unprocessed, tissue injection to the use of centrifugation protocols to concentrate and filter the BMA prior to injecting it in patients [47].

#### **4.1 Bone marrow harvesting needle systems**

Various bone marrow needle harvesting systems are available on the market, each with their own proprietary design characteristics and thus marrow aspiration dynamics when transferring marrow cavity cells through a needle system into collection syringes. In **Figure 2**, three different needle systems are shown. Potentially, different needle design features might affect the quality and viability of the harvested BM tissue, as well as the cellular yields. Therefore, BM needle system features and harvesting dynamics are important considerations when considering BMA procedures. Physicians have been using a variety of harvesting needles during the last decades, including the traditional Jamshidi™ harvesting needle (Ranfac Corporation, Avon, MA, USA). Based on design differences, not every BMA is born equal, and cellular yields, composition, and viability might vary among harvesting devices. For interpretation purposes, some of the cellular difference between two newly developed BMA needle harvesting systems, the Aspire Bone Marrow Harvesting System™ and the Marrow Cellution Bone Marrow Aspiration Device™ (EmCyte Corporation, Fort Myers, FL, USA, and Ranfac Corporation, Avon, MA, USA, respectively) is shown. A significant difference between the two harvesting

#### **Figure 2.**

*Bone marrow aspiration devices. (A) Jamshidi™ device (Ranfac Corporation, Avon, MA, USA), with a sharp and open distal tip, allows for more peripheral blood aspiration. (B) Aspire Bone Marrow Harvesting System™ (EmCyte Corporation, Fort Myers, FL, USA) consists of a separate trocar introducer and aspiration needle with a completely closed and blunt tip. The side holes of the aspiration needle are designed to minimize cellular trauma and hemolysis during aspiration. (C) Marrow Cellution Bone Marrow Aspiration Device™ (Ranfac Corporation, Avon, MA, USA) is used as an aspiration device only, to aspirate 10 ml of bone marrow, followed by unfiltered injection.*

systems is that the Marrow Cellution device is developed and used by physicians for BMA aspiration with direct injection only, without filtration or processing prior to patient injection [47]. Therefore, these specimens mimic the marrow cavity cellular content and their specific cell concentrations. This includes a red blood cell (RBC) content and hematocrit which is similar to the peripheral blood values. Conditional negative forces occur with the syringe pull during marrow aspiration; this particular BMA injectate can have high plasma-free hemoglobin (PFH) concentrations, which cannot be removed from the injectate. The Aspire™ harvesting system is designed to penetrate the trabecular bone, maintaining a quiescent tissue environment during deployment and collection, contributing to a reduction in tissue activation, plasma-free hemoglobin content, and clotting. The Aspire™ harvesting system is intended to provide a BMA for centrifugation processing, leading in this occasion to the creation of PurePRP SupraPhysiologic BMC® (EmCyte Corporation, Fort Myers, FL, USA). In **Table 1**, comparative laboratory data between the abovementioned needle systems in a bilateral patient harvesting model are disclosed.

**9**

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

**10 ml**

Hematocrit % 36.2 36.2 9.8

PFH mg/dl 913 721 299 Hemolysis % 4.6 3.2 1.6 Cell viability % 94.4 94.4 96.8 *BMA-MC, bone marrow aspirate Marrow Cellution System; BMA-A, bone marrow aspirate Aspire System; BMC, bone marrow concentrate; TNC, total nucleated cells; CD34+, hematopoietic stem cell marker/expression in bone marrow; CFU-F, colony-forming unit fibroblast: assay for bone marrow mesenchymal stem cell analysis, MSCs, mesenchymal stem cells. (BMA-MC, Marrow Cellution Device™—Ranfac Corporation, Avon, MA, USA; BMA-A,* 

*Aspire Bone Marrow Harvesting System™—EmCyte Corporation, Fort Myers, FL, USA).*

/mL 25.8 31.8 73.7

/mL 117 117 576

/mL 4.02 4.08 1.44

/mL 1.42 1.12 2.51

/mL 446 1.13 837

**BMA-A 10 ml**

**BMC 11 ml**

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

TNC × 106

RBCs × 109

**Table 1.**

Platelets × 106

CD34+ (HSCs) × 105

CFU-F (MSCs) × 103

**Laboratory parameters BMA-MC**

**4.2 Large vs. small BMA collection syringes**

*concentrate, in a bilateral patient model.*

**4.3 Image-guided aspiration**

reported anatomical site for BMA.

In theory, a larger-volume BMA collection syringe should produce a stronger negative pressure and therefore harvest more MSCs. However, Hernigou et al. found that the aspiration of only 10–20% of the full syringe volume resulted in a higher MSC concentration in both 10 and 50 ml syringes, indicating that high-quality harvesting of MSCs requires a significant negative pressure in the marrow cavity to liberate MSCs. They also concluded that the collection of MSCs decreased as the syringe was filled, at a lower negative pressure [40]. Therefore, smaller syringes and thus smaller aspiration volumes result in higher MSC concentrations than with larger aspiration volumes [48]. This translates to the physical equation, "Negative Pressure = Pull Force/Plunger Surface Area," resulting in the fact that with the same pull force and a smaller diameter syringe plunger, a higher negative pressure is created [49]. Lately, the authors use 10 ml syringes, employing a fast and intermittent pull technique to collect small volumes from different intra-trabecular sites (**Figure 3**). This is in accordance with a trend towards small-volume HPD aspiration techniques [50]. Another advantage for using 10 ml syringes is that anticoagulation protocols can be better managed. Smaller syringes fill considerably quicker than larger syringes, and smaller syringes can be adequately agitated with the anticoagulant to avoid clotting.

*Comparative quantification between two different bone marrow aspiration systems and bone marrow* 

In order to perform BM-MSC procedures, a certain volume and quality of marrow tissue are required in order to prepare a bone marrow concentrate (BMC). The aspiration volume is contingent on the processing volume of the BMC concentration system that is being used. It is imperative to locate precisely the donor site, as MSCs are located in the marrow cavity subcortical area and around the blood vessels [19]. The precise delivery of local anesthetics and safe trocar placement are accomplished by using image guidance during aspiration procedure. In the following section, we focus on the posterior super iliac spine (PSIS) sites, as it is the most frequently


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

*BMA-MC, bone marrow aspirate Marrow Cellution System; BMA-A, bone marrow aspirate Aspire System; BMC, bone marrow concentrate; TNC, total nucleated cells; CD34+, hematopoietic stem cell marker/expression in bone marrow; CFU-F, colony-forming unit fibroblast: assay for bone marrow mesenchymal stem cell analysis, MSCs, mesenchymal stem cells. (BMA-MC, Marrow Cellution Device™—Ranfac Corporation, Avon, MA, USA; BMA-A, Aspire Bone Marrow Harvesting System™—EmCyte Corporation, Fort Myers, FL, USA).*

#### **Table 1.**

*Regenerative Medicine*

**8**

**Figure 2.**

*followed by unfiltered injection.*

model are disclosed.

systems is that the Marrow Cellution device is developed and used by physicians for BMA aspiration with direct injection only, without filtration or processing prior to patient injection [47]. Therefore, these specimens mimic the marrow cavity cellular content and their specific cell concentrations. This includes a red blood cell (RBC) content and hematocrit which is similar to the peripheral blood values. Conditional negative forces occur with the syringe pull during marrow aspiration; this particular BMA injectate can have high plasma-free hemoglobin (PFH) concentrations, which cannot be removed from the injectate. The Aspire™ harvesting system is designed to penetrate the trabecular bone, maintaining a quiescent tissue environment during deployment and collection, contributing to a reduction in tissue activation, plasma-free hemoglobin content, and clotting. The Aspire™ harvesting system is intended to provide a BMA for centrifugation processing, leading in this occasion to the creation of PurePRP SupraPhysiologic BMC® (EmCyte Corporation, Fort Myers, FL, USA). In **Table 1**, comparative laboratory data between the abovementioned needle systems in a bilateral patient harvesting

*Bone marrow aspiration devices. (A) Jamshidi™ device (Ranfac Corporation, Avon, MA, USA), with a sharp and open distal tip, allows for more peripheral blood aspiration. (B) Aspire Bone Marrow Harvesting System™ (EmCyte Corporation, Fort Myers, FL, USA) consists of a separate trocar introducer and aspiration needle with a completely closed and blunt tip. The side holes of the aspiration needle are designed to minimize cellular trauma and hemolysis during aspiration. (C) Marrow Cellution Bone Marrow Aspiration Device™ (Ranfac Corporation, Avon, MA, USA) is used as an aspiration device only, to aspirate 10 ml of bone marrow,*  *Comparative quantification between two different bone marrow aspiration systems and bone marrow concentrate, in a bilateral patient model.*

#### **4.2 Large vs. small BMA collection syringes**

In theory, a larger-volume BMA collection syringe should produce a stronger negative pressure and therefore harvest more MSCs. However, Hernigou et al. found that the aspiration of only 10–20% of the full syringe volume resulted in a higher MSC concentration in both 10 and 50 ml syringes, indicating that high-quality harvesting of MSCs requires a significant negative pressure in the marrow cavity to liberate MSCs. They also concluded that the collection of MSCs decreased as the syringe was filled, at a lower negative pressure [40]. Therefore, smaller syringes and thus smaller aspiration volumes result in higher MSC concentrations than with larger aspiration volumes [48]. This translates to the physical equation, "Negative Pressure = Pull Force/Plunger Surface Area," resulting in the fact that with the same pull force and a smaller diameter syringe plunger, a higher negative pressure is created [49]. Lately, the authors use 10 ml syringes, employing a fast and intermittent pull technique to collect small volumes from different intra-trabecular sites (**Figure 3**). This is in accordance with a trend towards small-volume HPD aspiration techniques [50]. Another advantage for using 10 ml syringes is that anticoagulation protocols can be better managed. Smaller syringes fill considerably quicker than larger syringes, and smaller syringes can be adequately agitated with the anticoagulant to avoid clotting.

#### **4.3 Image-guided aspiration**

In order to perform BM-MSC procedures, a certain volume and quality of marrow tissue are required in order to prepare a bone marrow concentrate (BMC). The aspiration volume is contingent on the processing volume of the BMC concentration system that is being used. It is imperative to locate precisely the donor site, as MSCs are located in the marrow cavity subcortical area and around the blood vessels [19]. The precise delivery of local anesthetics and safe trocar placement are accomplished by using image guidance during aspiration procedure. In the following section, we focus on the posterior super iliac spine (PSIS) sites, as it is the most frequently reported anatomical site for BMA.

#### **Figure 3.**

*Bone marrow aspiration. After the aspiration needle has been advanced in the marrow cavity, the marrow is extracted using a firm, but a gentle, aspiration pressure is applied to the 10 ml syringe. The aspiration needle is easily rotated to collect marrow from a fresh area.*

#### *4.3.1 Ultrasound imaging*

When the PSIS is targeted, patients are positioned in the prone position, while avoiding lumbar lordosis. Sonographic assessment using a portable ultrasound system with a 5–2 MHz low-frequency curvilinear transducer is positioned in a transverse plane over the hyperechoic bilateral sacral cornua, with the patient lying prone and the monitor screen in the line of sight of the operator. The probe is then translated contralaterally from the physician, keeping the hyperechoic sacrum visualized. Next, the probe is translated proximally, with the hyperechoic ilium coming into view, while maintaining the hyperechoic sacrum, until the most superficial depth of the ilium is reached, known as the PSIS, contralateral to the examiner [51]. After identification of the PSIS, the most superficial depth is confirmed in both transverse and longitudinal orientation (**Figure 4**). With the probe in the transverse plane at the PSIS, the slope of the iliac wing is noted for correct angulation of the BM trocar, and the most superficial depth of the PSIS is brought under the most medial aspect of the ultrasound probe. Using a sterile marker, a mark and

**11**

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

directional line is made in both parallel and perpendicular orientations to form an intersection at the most superficial depth of the PSIS. This mark is maintained on the patient during skin preparation prior to the introduction off the BM trocar, and a superficial wheal of local anesthetic is placed at the point of planned trocar skin entry. Following the local antiseptic measures, sterile ultrasound gel is applied at the marked area, and a sterile probe cover is applied to the 5–2 MHz curvilinear array transducer. Typically, a mixture of local anesthetics is injected around the PSIS cortex and periosteal sleeve, under continued sonographic guidance, making sure to "walk off" the PSIS in four directions (superiorly, medially, laterally, and inferiorly), confirmed by sonographic guidance. The trocar is then introduced, using either a manual force that is perpendicular and slightly lateral to the patient, at 9–12 counterclockwise-clockwise rotations, or a mallet. The next steps of the procedure are subject to the implementation of the instructions for use provided by

*Ultrasound imaging of the PSIS. With the probe in the transverse plane, the PSIS is confirmed, and the slope (D) of the iliac wing is noted for correct angulation of the BM trocar (B), and the most superficial depth (C) of the PSIS is brought under the most medial aspect of the ultrasound probe. Note: (A) indicates the skin surface, and (E) marks the depth of the PSIS below the skin, in this patient approximately 1.6 cm (courtesy of* 

After proper patient positioning, the fluoroscopic equipment is installed to optimize the positioning for fluoroscopic imaging, using ipsilateral or contralateral oblique beam angulations for viewing the targeted PSIS site. The *perpendicular fluoroscopic approach* requires a beam angle around 15° ipsilateral to the PSIS entering laterally with angulation towards the sacroiliac joint. This angle will view the lateral ilium outer wall, and a needle is directed anteromedially. Fluoroscopic images support in positioning the tip of the trocar above the target area for entering the PSIS. The *parallel fluoroscopic approach* results in viewing down the PSIS table, at a 25° contralateral oblique beam position. This results in a classic view of the "teardrop" (**Figure 5**). Imaging can confirm the entry point into the PSIS table and visualize the angle through the cortex, allowing for safe trocar advancement in the BM cavity, at the tick part of the ilium bone [52]. Using proper fluoroscopic

the manufacturer of the aspiration harvesting system.

*4.3.2 Fluoroscopic imaging*

**Figure 4.**

*J. Rothenberg).*

*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*

#### **Figure 4.**

*Regenerative Medicine*

**10**

*4.3.1 Ultrasound imaging*

*easily rotated to collect marrow from a fresh area.*

**Figure 3.**

When the PSIS is targeted, patients are positioned in the prone position, while avoiding lumbar lordosis. Sonographic assessment using a portable ultrasound system with a 5–2 MHz low-frequency curvilinear transducer is positioned in a transverse plane over the hyperechoic bilateral sacral cornua, with the patient lying prone and the monitor screen in the line of sight of the operator. The probe is then translated contralaterally from the physician, keeping the hyperechoic sacrum visualized. Next, the probe is translated proximally, with the hyperechoic ilium coming into view, while maintaining the hyperechoic sacrum, until the most superficial depth of the ilium is reached, known as the PSIS, contralateral to the examiner [51]. After identification of the PSIS, the most superficial depth is confirmed in both transverse and longitudinal orientation (**Figure 4**). With the probe in the transverse plane at the PSIS, the slope of the iliac wing is noted for correct angulation of the BM trocar, and the most superficial depth of the PSIS is brought under the most medial aspect of the ultrasound probe. Using a sterile marker, a mark and

*Bone marrow aspiration. After the aspiration needle has been advanced in the marrow cavity, the marrow is extracted using a firm, but a gentle, aspiration pressure is applied to the 10 ml syringe. The aspiration needle is*  *Ultrasound imaging of the PSIS. With the probe in the transverse plane, the PSIS is confirmed, and the slope (D) of the iliac wing is noted for correct angulation of the BM trocar (B), and the most superficial depth (C) of the PSIS is brought under the most medial aspect of the ultrasound probe. Note: (A) indicates the skin surface, and (E) marks the depth of the PSIS below the skin, in this patient approximately 1.6 cm (courtesy of J. Rothenberg).*

directional line is made in both parallel and perpendicular orientations to form an intersection at the most superficial depth of the PSIS. This mark is maintained on the patient during skin preparation prior to the introduction off the BM trocar, and a superficial wheal of local anesthetic is placed at the point of planned trocar skin entry. Following the local antiseptic measures, sterile ultrasound gel is applied at the marked area, and a sterile probe cover is applied to the 5–2 MHz curvilinear array transducer. Typically, a mixture of local anesthetics is injected around the PSIS cortex and periosteal sleeve, under continued sonographic guidance, making sure to "walk off" the PSIS in four directions (superiorly, medially, laterally, and inferiorly), confirmed by sonographic guidance. The trocar is then introduced, using either a manual force that is perpendicular and slightly lateral to the patient, at 9–12 counterclockwise-clockwise rotations, or a mallet. The next steps of the procedure are subject to the implementation of the instructions for use provided by the manufacturer of the aspiration harvesting system.

#### *4.3.2 Fluoroscopic imaging*

After proper patient positioning, the fluoroscopic equipment is installed to optimize the positioning for fluoroscopic imaging, using ipsilateral or contralateral oblique beam angulations for viewing the targeted PSIS site. The *perpendicular fluoroscopic approach* requires a beam angle around 15° ipsilateral to the PSIS entering laterally with angulation towards the sacroiliac joint. This angle will view the lateral ilium outer wall, and a needle is directed anteromedially. Fluoroscopic images support in positioning the tip of the trocar above the target area for entering the PSIS. The *parallel fluoroscopic approach* results in viewing down the PSIS table, at a 25° contralateral oblique beam position. This results in a classic view of the "teardrop" (**Figure 5**). Imaging can confirm the entry point into the PSIS table and visualize the angle through the cortex, allowing for safe trocar advancement in the BM cavity, at the tick part of the ilium bone [52]. Using proper fluoroscopic

#### **Figure 5.**

*Fluoroscopy imaging of the PSIS. General prone position of the patient on a fluoroscopic table for BMA. The* parallel fluoroscopic approach *results in viewing down the PSIS table, at a 25° contralateral oblique beam position. This results in a classic view of the "teardrop," referring to the outline of the medial and lateral borders, as shown in the monitor. The tip of a needle (black circle), in the numbed skin, is marking the entry site of the bone marrow trocar to be placed in the marrow cavity, while the physician is on the ipsilateral side of the fluoroscope, viewing the correct position on the monitor (red circle) (courtesy of G. Flanagan II).*

techniques, the parallel approach technique allows for a safe deeper marrow penetration. However, at all times, regardless of the approach, avoid increased manipulation and tissue trauma using the sharp trocar, as this will increase the risk for neurovascular injury, bleeding, tearing of lateral gluteal muscle origins, and post-procedural pain.
