Introduction

As interest in the application of biologics to orthopaedic surgery grows, it is important to understand and optimize how we obtain and administer cell therapies. One such therapy, concentrated bone marrow aspirate (cBMA) is based on harvesting bone marrow progenitor cells with proliferative potential. Orthopaedic surgeons have begun to augment surgical procedures and treat degenerative conditions such as osteoarthritis and rotator cuff tears with bone marrow aspirate.^1–6 The use of cell therapies may contribute to decreased recovery time and morbidity rates.^7–9 Clinical application studies have suggested that success of these treatments is dependent on the number of cells harvested and used.^2,4 In a study involving tibial nonunions and a study involving rotator cuff repairs, improved healing rates were observed with greater proliferative potential of the cell product.^2,4Although bone marrow aspiration is frequently used for orthopaedic point of care applications, the number of progenitor cells harvested is variable and dependent on body mass, aspiration technique, and the location of harvest.^7,10 Understanding harvest site potential is a key step in the development of bone marrow aspirate for orthopaedic applications. The posterior superior iliac spine (PSIS) has outperformed the anterior iliac crest and appendicular osseous sites, when the greatest cellular yield of progenitor cells is preferred.^11,12 However, in arthroscopic rotator cuff surgery, there has been interest in using the proximal humerus as a source of progenitor cells to eliminate the need for a second surgical site. The purpose of this study is to determine whether cBMA from the PSIS or proximal humerus produces a more productive cellular harvest in patients undergoing arthroscopic rotator cuff repair. We hypothesize that viable stem cells can be harvested from both sites and that harvesting from the PSIS will result in greater cell counts than the proximal humerus.

Methods

Study protocols were approved by our institutional review board and consent forms were completed by all participants. Patients were prospectively enrolled in this study from January 2020 to March 2021. Inclusion criteria included patients presenting with small and medium sized rotator cuff tears who elected for arthroscopic repair. Exclusion criteria included patients who were equal to or greater than 80 years of age, those requiring superior capsular reconstruction or revision rotator cuff repair, and those with a medical history of diabetes, immune disorders, a previous metastatic or other cancer which required chemotherapy/radiation therapy, or rheumatoid arthritis. Additionally, any patient who had difficulty obtaining internet access, did not have an active e-mail address, or was unable to comprehend the study documents or give informed consent was also excluded.

Surgical Technique

All patients were in the lateral decubitus position on the operative room table for both bone marrow harvest procedures and arthroscopic rotator cuff repair. The PSIS harvest was performed first. The PSIS was palpated and outlined with a skin-marking pen, The skin over the PSIS was prepped with Chloraprep (BD, Franklin Lakes, NJ) and draped with Steri-Drape adhesive towel drapes (3M Healthcare, St Paul, MN). Although palpation, prepping and draping was not timed, anecdotally these steps took approximately 3 to 5 minutes.Lidocaine 1% was used to anesthetize the skin around the incision location. A stab incision was made in the skin over the PSIS. Two 30 cc syringes and a traditional 11-gauge, 11 cm Jamshidi needle (Fanfac Corporation, Avon, MA) were prerinsed with heparin. The two 30 mL syringes were loaded with 4 mL of anticoagulant citrate dextrose solution-A. The needle was advanced into the bone marrow cavity 3 to 4 cm. Bone marrow 30 mL was aspirated while the needle was withdrawn and rotated with one syringe. The needle was then reinserted divergently, and another 30 mL of bone marrow aspirated with the second syringe. This method has been described and quantitively studied with similar harvest to a multiple puncture technique.³ A sterile dressing was applied.The patients were then re-prepped and draped in standard fashion for shoulder arthroscopy in the lateral decubitus position. A diagnostic arthroscopy was performed, and the rotator cuff tear identified. While viewing from the standard posterior portal in the subacromial space, a spinal needle was used to localize the location and trajectory for placement of the first suture anchor for the rotator cuff repair. Once the placement of the proposed anterior suture anchor was confirmed, the 11-gauge bone marrow harvest needle was placed into this location and into the bone marrow cavity, approximately 2.5 to 3 cm. The arthroscopic fluid was then turned off, and 30 mL of bone marrow was aspirated while the needle was withdrawn and rotated. The needle was then reinserted divergently, and another 30 mL of bone marrow aspirated. The two 30 mL syringes were pre-filled with 4 mL of anticoagulant citrate dextrose solution-A. The bone marrow samples were then processed independently using the Arthrex Angel blood processing system (Arthrex, Naples, FL) using the 15% hematocrit setting with two separate disposables. One mL of cBMA from each harvest site was then removed and sent for analysis. Timing of bone marrow aspiration was performed and started with the first puncture for entry of the Jamshidi needle until completion of the bone marrow harvest.

Cell Analysis

Cellular composition was determined using an automated hemocytometer (Sysmex XE-5000). Complete blood count with differential and total nucleated cells (TNC) were obtained. TNC per milliliter was calculated. Proliferative potential was studied with colony-forming unit fibroblast (CFU) assays. Two hundred microliters of cBMA were placed into a 25 mL cell culture flask. Complete media 4 mL (a mixture of 445 mL of Dulbecco’s modified eagle medium low glucose, 50 mL of mesenchymal stem cell [MSC]-qualified fetal bovine serum, 5 mL of GlutaMax-I, and 250 µL Gentamicin) was added to each well, and the plate was incubated at 37°C. Standard cell culture conditions were used in a humidified atmosphere containing 5% CO₂ in air (~20% O₂). After 24 hours the plates were gently washed with phosphate buffered saline solution, and 4 mL media was added to remove nonadherent cells and leave only plastic adherent cells in the culture dish. The plates were returned to the incubator for 10 days. The plates were then removed, and cells were fixed in ice-cold methanol for 10 minutes at 4°C. The plates were stained with crystal violet, washed 4 times with water, and allowed to air dry. Colonies were then counted as long as more than 50 cells were present. Means, standard deviation (SD), and 95% confidence intervals (CI) were calculated. A dependent-samples T-test was used to interpret differences in total nucleated cells and CFU between the PSIS and proximal humerus. Correlations between demographic data (age, sex, body mass index [BMI]) and outcome data (total nucleated cell count and CFU) were analyzed with Pearson’s correlation coefficient and Spearman’s rho correlation as appropriate. Assumptions of normality were evaluated before the use of parametric tests. All analysis was performed using JASP (Version 0.14.1; University of Amsterdam, Amsterdam, Netherlands).The presence of stem cells from bone marrow is well established and was not the primary purpose of this study. This study’s purpose was simply to compare the cellular harvest between the humerus and iliac crest, which we determined would not require flow cytometry nor differentiation analysis.

Results

Twelve patients were prospectively recruited to participate in this study (7 male and 5 female). The average age of participants was 64.3 years (range 46.1-77.25 years) with BMI 26.8 (range 20.0-34.3). The average TNC count from the proximal humerus was 18.7 × 10⁶ cells/mL (95% CI, 4.4-33.0; SD, 24.8) with 3.9 CFU/mL (95% CI, 0.3-7.5; SD, 5.7). The average TNC count from the PSIS was 55.9 × 10⁶ cells/mL (95% CI, 25.3-86.4; SD, 52.9) with 32.5 CFU/mL (95% CI, 11.5-53.5; SD, 33.1). The PSIS had a greater TNC yield (P = .014) and CFU/mL (P = .024). Age and sex did not correlate with total cellular yield or CFU count. BMI correlated with CFU from the PSIS (r = −0.676, P = .032) but not from the humerus (r_s = 0.562, P = .091). Additionally, BMI correlated with TNC count from the humerus (r = 0.643, P = .028) but not from the PSIS (r = 0.183, P = .569). The average time to aspirate 60 mL of bone marrow aspirate was from the PSIS was 5.6 minutes and from the PH was 11.0 minutes) (P = .043).

Discussion

The hypothesis of this study was proven correct that cBMA harvested from the PSIS has, on average, a greater concentration of colony-forming progenitor cells and total nucleated cells than the proximal humerus. The conclusion is similar to previous studies, which have demonstrated increased cell counts in the iliac crest compared to appendicular harvest locations. Hyer et al.¹⁰ evaluated the viable nucleated cells and osteoblastic progenitor cell yield of 40 patients who underwent bone marrow aspiration from the anterior iliac crest, distal tibial metaphysis, and calcaneus. The study found the mean concentration of nucleated cells to be highest in the iliac crest (15.6 × 10⁶ cells/mL) when compared to the tibia (5.8 × 10⁶ cells/mL) and the calcaneus (7.1 × 10⁶ cells/mL). These findings paralleled the presence of osteoblastic progenitor cells with a significantly higher concentration in the anterior iliac crest (898.4 cells/mL) when compared to the tibia (32.4 cells/mL) and calcaneus (7.1 cells/mL).¹⁰ The results of our study demonstrate comparable findings with a 3.0-times increase in total nucleated cells and an 8.3-times increase in CFU from the PSIS compared to the proximal humerus. These results were similar to the findings from Narbona-Carceles et al.¹⁴ who compared the bone marrow aspirate harvested from the iliac crest, distal femur, and proximal tibia in 20 patients undergoing total knee arthroplasty. Cell aspiration from the anterior iliac crest had a sixfold greater concentration of mononucleated cells (10.05 × 10⁶ cells/mL) when compared to the distal femur (0.67 × 10⁶ cells/mL) and proximal tibia (1.70 × 10⁶ cells/mL).¹⁴ Studies have consistently shown the iliac crest to have the highest concentration of osteoblastic progenitor cells.

Similar to other appendicular harvest sites, bone marrow aspirate from the proximal humerus has consistently demonstrated the presence of osteoblastic progenitor cells. Previous studies have investigated the proximal humerus potential for osteoblastic progenitor cells when compared to other locations in different subjects. Beitzel et al.¹⁵ evaluated the bone marrow aspirate from the proximal humerus in 55 patients and the distal femur in 29 patients undergoing shoulder and knee arthroscopy, respectively. The nucleated cellular yield of the proximal humerus was 38.7 × 10⁶ cells/mL with 883.9 MSC/mL.¹⁵ Similarly, the distal femur had a concentration of 25.9 nucleated cells/mL and 551.3 MSC/mL.¹⁵ Unfortunately, the study did not compare the yield in the same patients, which limits the ability of the study to compare the potential of each harvest site. However, the study did demonstrate the presence of viable progenitor cells in the proximal humerus in patients undergoing shoulder and knee arthroscopy. Similarly, Otto et al.¹⁶ evaluated the cellular yield from the proximal humerus in 87 patients undergoing shoulder arthroscopy and from the ilium in 30 patients undergoing hip arthroscopy. The study found a yield of 27.07 × 10⁶ nucleated cells/mL and 1516.62 CFU/mL of bone marrow aspirated from the proximal humerus.¹⁶ In contrast, the ilium yielded 24.01 × 10⁶ nucleated cells/mL with 979.17 CFU/mL of bone marrow aspirate.¹⁶ The concentration of cells was greater in the humerus than the ilium; however, the samples were obtained in different patients, which limits comparison. In addition, the site used for bone marrow aspiration from the ilium was at the level of or just proximal to the acetabular sourcil, which may not be equivalent to the anterior superior iliac spine (ASIS) or PSIS in terms of cellular yield. A study by Pierini et al.¹¹ evaluated different aspiration sites in the ilium to determine if the PSIS or ASIS had a greater cellular yield. Bone marrow aspirate from the ASIS and PSIS were analyzed from 22 patients. The mononucleated cell yield was similar between the 2 harvest sites; however, the mean yield of progenitor cells was found to be significantly higher in the PSIS (269.3 cells/10⁶ nucleated cells) when compared to the ASIS (166.4 cells/10⁶ nucleated cells).¹¹ These results reinforced the PSIS as the “gold standard” harvest location for obtaining bone marrow aspirate. The results of our study confirm the PSIS as a more potent site for harvesting bone marrow than the proximal humerus. Additionally, our study also confirms the findings from previous studies that the proximal humerus is a viable location for obtaining progenitor cells. Comparing CFU counts between sites and studies produces confusion because culturing protocols, including the number of media changes, and culturing experiences differ between institutions and studies. Variation in culturing protocols is a limitation when one compares studies.

One interesting finding in this study is that the time required to aspirate large volumes of bone marrow aspirate from the proximal humerus took longer than the time needed to aspirate from the PSIS (5.6 vs 11.0 minutes). The time to aspirate did not take into account the additional time that it would take to prep and drape the PSIS, while the PH is already prepped into the field and does not require extra time. Anecdotally, we have found that it takes 3 to 5 minutes to palpate the PSIS, prepare with a surgical prep, and drape out with towels before PSIS harvest. We did find that the PSIS allowed for a much easier flow of bone marrow contents into the syringe compared with the proximal humerus. This may be a result of an easier and more direct angle to access the medullary canal in the PSIS, the larger size of the posterior ilium in relation to the proximal humerus, a result of gravity, or dependent on surgeon and experience. A previous study by Otto et al.¹⁶ used a standardized 60 seconds of suction on the syringe to aspirate the bone marrow of the proximal humerus and ilium in patients undergoing shoulder and hip arthroscopy, respectively. The authors were able to aspirate 91.67 mL of bone marrow aspirate from the ilium and 85.63 mL from the proximal humerus during that time.¹⁶ This study demonstrated that large volumes of aspiration from both sites can be achieved quickly. The difference in time in our study may be partially attributed to a difference in how the procedure was timed. Our study calculated the time for the entire aspiration from start to finish, whereas Otto et al.¹⁶ reported the amount of time the syringe was suctioning. Both studies do, however, confirm that large volumes of bone marrow can be aspirated rapidly in the operating room from both the proximal humerus and ilium.

This study consists primarily of data collected in the laboratory setting, determining the cellular yields of cBMA from the proximal humerus and PSIS. Previous studies have indicated that the number of MSC is important in seeing a clinical benefit. Hernigou et al.² performed a study to determine whether cBMA injections after rotator cuff repair enhanced healing rates when compared to rotator cuff repair alone. The study found an increased rate of intact rotator cuff repairs (87% vs 44%) at 10-year follow-up in patients who had their repairs augmented with cBMA. Additionally, the study found that the patients with retears had a lower number of transplanted MSCs (1500 cells/mL vs 4200 cells/mL). However, because there is great variation in harvest and culture protocols, it is difficult to determine the ideal concentration or number of MSC needed to provide a clinical benefit until methods are standardized. TNC can be used as an indication of the cellular yield of a harvest, whereas the CFU per mL provides the concentration of MSC per mL in the cBMA. Both measurements together can give the reader an idea of the total number of MSC injected. However, future studies are still needed to establish the optimal cellular concentration, number of MSC, and volume of cBMA needed to enhance healing potential.

Limitations

One limitation of this study is the lack of flow cytometry and cell differentiation data. The small sample size of this study and lack of a prior power calculation are limitations as well, which does raise the possibility of a type I alpha error. However, because our data are in line with the conclusions from previous studies, we do believe this to be unlikely. Subject enrollment was limited by funding constraints.

Conclusions

The cBMA harvested from the PSIS resulted in a 3.0 times greater cellular yield and an 8.3 times greater proliferative product than cBMA from the proximal humerus.

References

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