The effect of a single consecutive volume aspiration on concentrated bone marrow from the proximal humerus for clinical application

Biologic cell-based therapy has garnered increasing attention over the past two decades [1‐3], with recent research attempting to optimize harvest and processing methods to increase the number of pluripotent stem cells [4, 5]. In the field of orthopaedics, various sources have been examined in order to obtain these progenitor cells, however, bone marrow aspirate is the current gold standard [6].

Although various locations may be used to obtain bone marrow, the quality and effectiveness of an aspirate still depends on the concentration of connective tissue progenitors (CTPs) within the sample [7]. According to Muschler et al., this concentration is influenced by the volume of aspirate [8]. The authors found that smaller samples may contain a higher concentration due to larger volumes becoming diluted with peripheral blood [8]. The authors compared one-milliliter, two-milliliter and four-milliliter aspirates obtained from the anterior iliac crest [8]. They found that using one-milliliter aspirates four times would provide almost twice the amount of colony forming units (CFUs) compared to one four-milliliter aspirate [8]. Therefore, aspiration techniques using multiple aspirations of smaller volumes in order to limit dilution with peripheral blood would ensure a higher concentration of CTPs [5, 8, 9].

Preventing dilution of a bone marrow aspirate is of clinical concern, as effectiveness of bone marrow aspirate (BMA) applications depends on the total number of CTPs in the sample [10]. With the percentage of mesenchymal stem cells comprising as little as 0.01% of a bone marrow aspirate, the process of centrifugation to increase the nucleated cell count has become of clinical importance [11]. Using a centrifuge, cellular components of the sample can be separated, allowing for generating a highly concentrated BMA (cBMA) sample regardless of prior dilution with peripheral blood. Mazzocca et al. first described an arthroscopic technique for obtaining bone marrow aspirate from the proximal humerus [4]. This technique allows the harvest site to later be used for placement of the medial anchor during rotator cuff surgery, thus decreasing donor site morbidity [4]. Using this method, a previous study by Voss et al. found that CTPs could be present even after 40 ml of bone marrow aspiration from the same harvest site [12]. However, their study was limited in its focus on aspiration technique, and did not evaluate patient factors or the sequential decrease in CFUs with each aliquot obtained [12].

The purpose of the current study was to investigate the effect of four consecutive aliquots from a single high-volume aspiration on the number of nucleated cells and CFUs in concentrated bone marrow aspirate taken from the proximal humerus. Age and sex were evaluated as secondary outcome measures. The authors hypothesized that there would be continued presence of nucleated cells and CFUs even after the fourth aliquot, with no significant differences between the first and second 10 cc aliquot. The secondary hypothesis was that increasing age would result in a significant decrease in the number of nucleated cells and CFUs across the four aliquots.

The most important finding of this study is that despite significantly less nucleated cells and CFUs per sequential aliquot, connective tissue progenitor cells were still found to be present after aspiration of 40 ml of bone marrow from the proximal humerus. In addition, when comparing the mean number of NCs or CFUs in the first aliquot to the sum of the averages of the second, third, and fourth aliquot, there was no significant difference. Increasing age of the patient, resulted in no significant decrease in the number of NCs and CFUs across the four consecutive aliquots.

In order to avoid hemodilution, several studies have supported the use of repeated smaller aspirates, ranging from 1 to 4 ml [8, 14, 15]. However, multiple small aspirations form a single trocar or from various sites may lead to longer surgical time, risk of infection or damage to nearby neurovascular structures [16]. Hernigou et al. examined the effect of syringe size on mensenchymal stem cell (MSC) aspiration, and found that aspirating only part of the tube’s full capacity (10–20%) produced a higher MSC concentration than aspirating either a 10 mL or 50 mL tube to completion [17]. However, their study focused on single aspiration from multiple sites of the iliac crest, rather than the cumulative effects of fractioning samples from a single aspirate [17].

Voss et al. found that using a non-fenestrated trocar yielded significantly higher cell counts, which was the technique performed in this study [12]. While they demonstrated continued prevalence of CTPs with sequential aspiration, they were limited in number of patients as well as the influence of fractionated layer volume following centrifugation [12]. We found that each aliquot had similar amounts of hemodilution with no effect on the final number of nucleated cells and CFUs in the sample. In addition, we demonstrated that the sum of CFUs from the sequential aliquots yielded similar progenitor cells compared to the initial 10 cc of aspiration. This is of clinical importance, as efficacy of mesenchymal stem cells has direct correlation with the quantity of these cells [18]. This study also highlights the importance of age on the number of CFUs and nucleated cells within the first fractioned aliquot, as well as its impact on subsequent concentrated aliquots. This supports previous findings that the number of osteoprogenitor cells in bone marrow demonstrates marked decrease after the first two decades [19].

The efficacy of autologous bone marrow for clinical applications depends on the concentration of necessary progenitor cells [10]. While certain patient characteristics, such as alcohol abuse [20] and smoking [21] can negatively affect bone marrow aspirate quality, optimizing surgical technique is essential for success of treatment. Although aspiration of bone marrow from the iliac crest is still considered the gold standard [5, 22‐24], complications such as hematoma and nerve palsy have been reported [25]. While the proximity of the axillary nerve and artery make the proximal humerus amenable to similar risks, the ability to obtain the sample under direct visualization during rotator cuff repair makes this an ideal location. Mazzocca et al. first described the proximal humerus as a more desirable source of MSCs for rotator cuff repair due to its ease of attainment [4]. Along with attainment of these cells, quantifying their type and characteristics has varied in the literature. The International Society of Cell Therapy defined adult stem cells that adhere to culture plastic and express markers such as CD73, CD90, and CD105 [13]. Juneja et al. demonstrated a viable method for aspiration of mesenchymal stem cells from the femur of patients undergoing total hip and knee arthroplasty [26]. Using flow cytometry, the authors demonstrated that cells obtained were positive for several markers (C73, CD105, CD90), meeting the criteria for stem cell criteria [26]. In this study, colony-forming units was used as a measurement for the number of connective tissue progenitor cells. As analysis of CFUs may not be ideal intraoperatively, El-Jawhari recently proposed a novel rapid flow cytometry assay that can be used to quantify bone marrow-derived MSCs within an intraoperative time frame [27].

There are several limitations to this study. This is an in-vitro study without direct evidence of the effects of these implanted cells. In addition, nucleated cell count and the CFU assay used in this study do not give information about the heterogeneity of the CTPs and colonies they form [28]. Another limitation is the wide inter-individual variability with regards to counting the number of nucleated cells and CFUs, making comparisons to prior studies difficult [8, 22, 26, 29]. This includes disparities as to when a CFU should be counted (e.g., 7, 10, and 14 days), as well as what should be the minimum number of aggregated cells used to define a CFU, which has been shown to have gross variability (e.g., 8 and 50) [26, 30, 31]. Accordingly, counting small colonies may generate higher total numbers compared to studies in which a colony was defined as a cluster of significantly more cells, which may lead to misinterpretation of the data. In addition, these methods are dependent on the expertise in microscopic evaluation of those examining [26, 30]. In this study, an experienced research associate (MBM) performed the colony counting and a CFU was defined to be a cluster of 8 or more cells [13]. However, counting CFUs may underestimate the actual number of CTPs as more than one CTP can give rise to a colony [28]. Additionally, this study had no comparison group to evaluate if the findings are consistent with aspiration of other sites, such as the iliac crest or vertebral body.

In conclusion, while the initial aliquot provided the greatest number of nucleated cells and cultured CFUs, the addition of each sequential volume aspirate yielded similar amounts in total. This demonstrates the potential effectiveness of obtaining of higher volume aspirates from the proximal humerus during rotator cuff repair.