BMA and BMACs, which contain a complex mix of nucleated cells, platelets and growth factors, are known to aid in the healing of a variety of orthopedic injuries and to promote angiogenesis in ischemic tissue [2, 19,20,21,22]. While BMA and BMACs show significant promise in the clinic, it is important to validate individual bone marrow preparations given the numerous methods for aspirating and concentrating bone marrow currently in use [11] as well as the different locations of stem cells within the marrow space [13, 14]. This is particularly important given that aspirates with higher levels of CFU-f cells and/or CD34+ cells have been shown in a variety of studies to correlate with improved patient outcomes [1,2,3,4, 21].
Given the association between higher levels of stem/progenitor cells and positive patient outcomes, verifying that BMAs and BMACs contain sufficient numbers of these cells is of clinical relevance. In this study, we found that the MC system which employs the SSLM method, produces ~ 10 mL aspirate with high concentrations of CFU-fs and/or CD34+ cells. Most traditional needle aspirates are sourced from a cannula with an open distal end without a mechanical means to precisely reposition the cannula. These traditional needle techniques normally produce aspirates that contain between 200 and 300 CFU-f/mL [11]. In the three direct comparisons with three different clinicians and two different centrifuge systems, the MC system had significantly higher amounts of CFU-fs and tended toward similar or higher numbers of CD34+ and CD117+ cells as compared to centrifugation systems (Tables 1, 2, 3). The only exception was the significantly higher levels of CD34+ cells in the first comparison where the BMAC draw was performed using smaller aspirations at three different insertion sites, as opposed to a single large aspiration at only one insertion site as performed in the other two comparisons. Despite the elevated CD34+ cells in this BMAC, the MC system aspirate still had significantly higher CFU-f levels. Interestingly, the smaller volume aspirations done at the three different insertion sites for the BMAC draw seemed to preferentially increase the number of CD34+ cells without a corresponding increase in CFU-f levels. Previous studies have shown while the CFU-fs are predominately found in the CD34+ population, only a small fraction of CD34+ cells are capable of forming CFU-fs so the levels may not always correlate [23].
There is increasing evidence that different MSC populations are found in unique niches within the bone. MSC populations have been identified within the cortical bone space and trabecular bone cavities [12, 24], as well as within endosteal and perivascular niches within the marrow space [13, 25]. It is possible that different aspiration techniques can preferentially target these subpopulations. The BMAC aspirates were done using an aspiration needle with an open-ended cannula that has five side ports but that draws preferentially through the largest distal open end that is oriented toward the center of the bone space. In contrast, the MC system draws only from the sides and not the center as the distal opening is closed. Given these differences, the endosteal population near the inner cortical bone space [13], located adjacent to the side ports in the MC system, may be preferentially targeted with the SSLM method. As a result, while multiple small incisions/draws using a traditional needle may increase the CD34+ count, this technique may target a cell population from the inner marrow space that is not as rich in CFU-f forming cells. Our data supports the hypothesis that a different cell population richer in MSCs may reside along the endosteal niche near the cortical surface of the bone, a population that can be preferentially targeted based upon the aspiration method.
High concentrations of CFU-fs were found utilizing the MC system by two different clinicians using the analysis of two different independent laboratories. In the case series, the MC system produced 10 mL aspirates that had an average of 2885 CFU-f/mL. The total number of CFU-fs found in the MC system aspirates, roughly 28,000, represents a level of progenitor/stem cells that is in line with concentrations from other studies that have shown therapeutic success in reducing disc pain [26], treating hip osteonecrosis [27] and ameliorating cartilage defects [1]. In addition, the number of CFU-fs/mL found here in the case series was greater or similar than those reported previously for centrifuge-based final products [11, 21].
Overall, the data presented here challenges the rationale for aspirating large volumes of bone marrow and then volume reducing through centrifugation. While centrifugation systems allow for higher TNC levels as seen here, they did not in general produce BMACs with higher CFU-f, CD34+ or CD117+ concentrations than the MC system. The CFU-f/mL yields in the BMACs tested here are at the low end of the range as compared with previous BMAC studies that found CFU-f/mL yields in the 500–3000 range [11, 16, 21, 26, 28]. The high end of this range is similar to what was found here with the MC system case series. Based upon the CFU-f, CD34+ and CD117+ levels, the comparison data suggest that higher concentrations of HSCs and MSCs are found following the SSLM method [29].
There are several likely explanations for the high levels of stem/progenitor cells harvested with the SSLM method. First, the MC system automatically repositions the aspiration cannula allowing it to mimic multiple puncture sites each with small 1 mL aspirations. Previous studies have shown that large volume aspirates (more than 2 mL) from a single site using traditional needles tend to be infiltrated by significant amounts of peripheral blood, which contains very few MSCs and HSCs and leads to lower CFU-f and CD34+ cell counts [9, 10]. This is particularly important in diabetic, atherosclerotic and elderly patients in which the amount of MSC and HSC is significantly reduced [30,31,32]. Secondly, the MC system aspirates from side ports, thereby (1) pulling in fluid from across a greater geography of the marrow space and (2) targeting the endosteal niche closest to the inner cortical plate, a region known to be rich in stem cells [13, 25]. While enzymatic digestion is necessary to fully harvest MSCs from this site, aspiration of marrow from this region appears to yield more MSCs than marrow harvested from traditional needle systems. Finally, the inefficiencies of the centrifugation process can leave significant numbers of stem/progenitor cells behind in the discarded portion of the processed marrow. Studies have shown that many MSCs are present as aggregates in bone marrow aspirates, aggregates that can be removed by the filtering and centrifugation steps associated with BMAC preps [33, 34].
In addition, very small embryonic-like stem cells (VSELs), which are found in BM and can contribute to tissue regeneration, are lost during common centrifugation protocols due to their high nucleo-cytoplasmic ratio [35, 36]. Given the variety of cells in the bone marrow that can aid in tissue regeneration (HSCs, MSCs, VSELs) [33, 37, 38] and the fact that significant amounts of these cells can be lost in the centrifugation process, the ability to avoid centrifugation can be a significant benefit in preparing a robust biologic.
In order to better characterize the aspirates generated by the MC system, we examined the correlation between TNC and CFU-f counts obtained from MC system aspirates. It was found that the two values did show a small but highly significant correlation with higher TNC values associated with higher CFU-f counts. Previous studies using BMAC have found a similar correlation [28]. In addition, we found a small but significant correlation between age and CFU-f counts using this process, with aspirates from older individuals correlating with lower CFU-f yields. These results are in general agreement with previous research [39].
While the data presented here demonstrates that following the SSLM method produces an aspirate that contains high CFU-f and CD34+ levels, there are limitations associated with the study. First, while suitable for a pilot study, the sample size for each of the three comparison studies was small. Second, the different methodologies at the three labs were not standardized. However, the fact that CFU-f values were comparable between the two labs that tested CFU-fs suggests that the differences in protocols did not substantially affect the outcome of the study. Despite this, future studies should include a larger sample of side-by-side comparisons with standard laboratory protocols in place. Given the possibility that different aspiration techniques can preferentially target different bone marrow niches, further studies investigating the geographical location of different cell types within the marrow space and the influence of different aspiration protocols on selecting these cells is warranted. Finally, given that higher numbers of MSCs as measured by CFU-fs have been associated with positive treatment outcomes, future studies should include follow-up on patients treated with these aspirates.