Abstract
Objective
The purpose of this study was to determine the M1/M2 macrophage ratio in concentrated bone marrow aspirate (cBMA) in patients undergoing surgical intervention augmented with cBMA for osteochondral lesions of the talus (OLTs).
Design
Samples of peripheral blood (PB), bone marrow aspirate (BMA), and cBMA were collected during the procedure. The samples were analyzed by automated cell counting and multicolor fluorescence-activated cell sorting with specific antibodies recognizing monocytes (CD14+ CD16+) and the M1 (CD86+) and M2 (CD163+CD206+) populations within that monocyte population. Cytokine concentrations within the samples were evaluated with enzyme-linked immunosorbent assay (ELISA). The composition of cBMA was compared between 2 commercially available BMA concentration systems.
Results
Thirty-eight patients with a mean age of 43.2 ± 10.1 years old undergoing a surgical procedure for the treatment of OLTs involving the use of cBMA were included. cBMA had a mean fold increase of 4.7 for all white blood cells, 6.1 for monocytes, 7.9 for lymphocytes, 2.4 for neutrophils, and 9.6 for platelets when compared to BMA. The mean M1/M2 ratio for PB, BMA, and cBMA was 15.2 ± 12.0, 20.8 ± 13.3, and 22.1 ± 16.0, respectively. There was a statistically significant higher concentration of interleukin-1 receptor antagonist (IL-1Ra) in the cBMA sample (8243.3 ± 14,837.4 pg/mL) compared to both BMA (3143.0 ± 2218.5 pg/mL) and PB (1847.5 ± 1520.4 pg/mL) samples. The IL-1Ra/IL-1β ratio for PB, BMA, and cBMA was 790.6 ± 581.9, 764.7 ± 675.2, and 235.7 ± 192.1, respectively. There was no difference in the cBMA M1/M2 ratio (19.0 ± 11.1 vs 24.0 ± 18.3) between the Magellan (Isto Biologics, Hopkinton, Massachusetts) and Angel systems (Arthrex Inc, Naples, Florida).
Conclusion
This prospective study found that the M1/M2 ratio in cBMA was 22.1 ± 16.0, with significant patient to patient variation observed. Overall, there was no statistically significant difference in the M1/M2 ratio across PB, BMA, and cBMA samples. This is the first study to characterize the macrophage subpopulation within cBMA, which may have significant clinical implications in future studies.
Keywords: concentrated bone marrow aspirate, BMAC, macrophage, M1/M2 ratio
Introduction
Osteochondral lesions of the talus (OLT) are a challenging pathology to treat due to the poor regenerative capacity of the native cartilage. 1 This can primarily be attributed to the avascular nature of the hyaline cartilage that lines the talar dome. 2 Novel treatment strategies have been developed in an effort to enhance the biology of the local microenvironment, including concentrated bone marrow aspirate (cBMA).1,3 cBMA is produced following centrifugation of bone marrow aspirate (BMA), which can be harvested from a range of donor sites, the most common of which is the iliac crest. 4 The resulting autologous product contains a host of growth factors, anti-inflammatory proteins such as interleukin-1 receptor antagonist (IL-1Ra) and a small proportion of mesenchymal stem cells (MSCs) (<1% of all stromal cells), with variations in cellular composition observed between different BMA concentration systems.5,6 Although the precise mechanism of action of cBMA is yet to be fully elucidated, it is hypothesized that its trophic, anti-inflammatory, and immunomodulatory properties contribute to the promotion of cartilage repair. 5
In recent years, there has been a growing interest in the role of macrophages in both the progression and repair of cartilage injuries. 7 Simplistically, monocytes have the ability to polarize into either pro-inflammatory, microbicidal M1 macrophages or, anti-inflammatory M2 macrophages. 7 M1 macrophages, activated by stimuli such as interferon-gamma (IFN-γ) and lipopolysaccharide (LPS), induce cartilage damage by upregulating genes associated with matrix degradation, secretion of pro-inflammatory cytokines, including interleukin-6 (IL-6), IL-1β, tumor necrosis factor alpha (TNF-α), and increasing the production of proteolytic enzymes such as matrix metalloproteinases and aggrecanases. 7 Conversely, M2 macrophages, induced by cytokines such as IL-4, IL-13, or IL-10, facilitate cartilage repair by promoting an anti-inflammatory microenvironment, via secretion of factors such as IL-10 and IL-1RA. 7 In addition, M2 macrophages release pro-chondrogenic factors such as transforming growth factor beta (TGF-β), insulin-like growth factor-1 (IGF-1), and IGF-2, supporting chondrogenesis and aiding in the restoration of cartilage injuries. 7 The delicate balance between M1 and M2 macrophage subpopulations within the joint microenvironment is a potential therapeutic target in the treatment of cartilage lesions, with a lower or more favorable M1/M2 ratio possibly associated with enhanced cartilage repair. 7 cBMA has been demonstrated to effectively augment the reparative process for cartilage lesions and is a source of a high concentration of macrophages with a potentially favorable M1/M2 ratio. 5 However, to date, the M1/M2 ratio has not been described in cBMA.
Thus, the purpose of this prospective study was to determine the M1/M2 ratio in cBMA in patients undergoing surgical intervention augmented with cBMA for OLTs. In addition, we sought to provide a comprehensive evaluation of the cellular and molecular composition of cBMA compared to BMA and peripheral blood (PB). Finally, we attempted to compare the composition of cBMA produced by 2 commercially available BMA concentration systems.
Methods
Patient Recruitment
This prospective study was conducted between March 1, 2021 and December 1, 2022 following Institutional Review Board approval (i21-00163). The inclusion and exclusion criteria are listed in Table 1 . Inclusion criteria consisted of patients undergoing a surgical procedure for the treatment of an OLT between the ages of 18 and 79. Exclusion criteria included medical history significant for systemic inflammatory disorders, corticosteroid use, chronic antiplatelet use, malignancy, diabetes, previous cBMA injections in the 6 months prior to their procedure and bacterial, viral or fungal infection in the 3 months prior to their procedure. All procedures were performed by the same senior attending foot and ankle orthopedic surgeon.
Table 1.
Inclusion and Exclusion Criteria.
| Inclusion Criteria | Exclusion Criteria |
|---|---|
| Patients receiving concentrated bone marrow aspirate as part of their treatment for an osteochondral lesion of the talus | History of systemic inflammatory disorders |
| Age > 18 | History of diabetes |
| Age < 80 | History of malignancy |
| Chronic corticosteroid use | |
| Chronic anti-platelet use | |
| Received an injection of concentrated bone marrow aspirate in the previous 6 months | |
| History of bacterial, viral or fungal infection within the previous 3 months |
Blood and BMA Collection
One mL of PB was obtained via venepuncture prior to commencement of the procedure. Bone marrow was aspirated from the iliac crest into a 30-mL syringe containing 4 mL Anticoagulant Citrate Dextrose (ACD). The needle was advanced 1 cm and rotated 90° after each 5 mL aspirate until a total of 60 mL was aspirated in 2 syringes. One mL was retained as the BMA sample for the study, and the remainder was processed in 2 systems; Angel (Arthrex Inc, Naples, Florida) and Magellan (Isto Biologics, Hopkinton, Massachusetts). All aspirations were performed by the same senior attending foot and ankle orthopedic surgeon. Representatives from both cBMA companies observed aspiration and concentration techniques. Samples were shipped deidentified to prevent bias in sample processing and analysis. All samples were processed within 24 h of collection.
Cytology
Complete blood counts were performed on PB, BMA, and cBMA using an Element HT5 hematology analyzer (HESKA, Loveland, CO) to assess platelet and white blood cell counts, including monocytes, lymphocytes, and neutrophils.
Flow Cytometry
Mononuclear cells were isolated from PB, BMA and cBMA using Ficoll-Paque PREMIUM density-gradient centrifugation according to the manufacturer directions (GE Healthcare, Chicago, IL) and then treated with RBC Lysis Buffer (BD, Franklin Lakes, NJ) to remove remaining red blood cells. Cells were labeled with fluorescent antibodies CD3-FITC (Invitrogen, Carlsbad, CA) to identify T cells, CD14-BUV737 and CD16-BUV563 (BD) to identify monocytes, CD80-PE-Cy7 (BD) for M1 monocytes, and CD163-BV480 and CD206-BV750 (BD) to identify M2 monocytes.
Analysis for T cells (CD3) was performed by gating for lymphocytes, while analysis for monocytes (CD14, CD16) and the M1 (CD80) and M2 (CD163, CD206) subsets was performed by gating for monocytes. Both initial gating strategies were followed by a forward scatter, then side scatter doublet exclusion. All gates for fluorescent markers were determined by unstained and Fluorescence Minus One (FMO) controls. Flow analysis was performed on a BD FACSymphony A5 Cell Analyzer (BD), and analyzed using FlowJo software (TreeStar Inc, Ashland, OR).
IL-1Ra and IL-1β Assays
Multiplex assays were performed according to manufacturer directions to measure IL-1β and IL-1Ra, using the Luminex 200 instrument (Luminex Corp., Austin, TX).
Statistical Analysis
Cell populations in PB, BMA, and cBMA were analyzed using a least squares model with horse as random effect. Tukey’s post-hoc was used with P values ≤.05 were considered significant. To compare cell populations between systems, a Wilcoxon rank sum test was used with a 1-way chi-squares test with P values ≤.05 were considered significant. Statistical analyses were performed using JMP Pro 15 (SAS Institute, Cary, NC).
Results
Patient Demographics
Patient demographics are listed in Table 2 . In total, 38 patients (38 ankles) were included in the final analysis. There were 22 males (57.9%) and 16 females (42.1%). The mean age at the time of surgery was 43.2 ± 10.1 (range, 20-68) years and the mean body mass index was 27.1 ± 3.2 kg/m2 (range: 21.2-30.4 kg/m2). All patients received cBMA as part of their treatment for OLT. No complications associated with collection of the PB nor BMA were observed.
Table 2.
Patient Demographics.
| N patients | 38 |
| M/F | 22/16 |
| Mean age (y) | 43.2 ± 10.1 |
| Mean BMI (kg/m2) | 27.1 ± 3.2 |
| Mean lesion size (mm2) | 87.4 ± 8.9 |
N = number; M/F = male/female; y = years; BMI = body mass index.
Cytology and Platelet Concentration
The concentration of total white blood cells (WBCs) and all subtypes of WBCs were greater in cBMA compared to PB ( Table 3 ). There was an 11.3-fold increase in WBC concentration in cBMA compared to PB. There was a 6.2-fold increase in neutrophil concentration in cBMA compared to PB. There was a 16.1-fold increase in monocyte concentration in cBMA compared to PB. There was a 16.5-fold increase in lymphocyte concentration in cBMA compared to PB. There was a 7.3-fold increase in platelet concentration in cBMA compared to PB.
Table 3.
Cytology and Platelet Concentrations in Peripheral Blood, Bone Marrow Aspirate, and Concentrated Bone Marrow Aspirate.
| PB | BMA | cBMA | |
|---|---|---|---|
| WBC (1000/µL) | 4.7 ± 1.3 | 12.2 ± 7.2 | 54.1 ± 39.3 |
| Neutrophils (1000/µL) | 2.6 ± 1.2 | 6.7 ± 4.9 | 16.2 ± 14.6 |
| Monocytes (1000/µL) | 0.4 ± 0.2 | 1.1 ± 0.7 | 6.9 ± 6.6 |
| Lymphocytes (1000/µL) | 1.4 ± 0.4 | 2.9 ± 1.4 | 22.5 ± 15.4 |
| Platelets (1000/µL) | 164.3 ± 42.4 | 123.9 ± 46.6 | 1192.8 ± 717.5 |
PB = peripheral blood; BMA = bone marrow aspirate; cBMA = concentrated bone marrow aspirate; WBC = white blood cell.
The concentration of total WBCs and all subtypes of WBCs were greater in cBMA compared to BMA. There was a 4.7-fold increase in WBC concentration in cBMA compared to BMA. There was a 2.4-fold increase in neutrophil concentration in cBMA compared to BMA. There was a 6.1-fold increase in monocyte concentration in cBMA compared to BMA. There was a 7.9-fold increase in lymphocyte concentration in cBMA compared to BMA. There was a 9.6-fold increase in platelet concentration in cBMA compared to BMA.
The concentration of total WBCs and all subtypes of WBCs were greater in BMA compared to PB. There was a 2.7-fold increase in WBC concentration in BMA compared to PB. There was a 2.6-fold increase in neutrophil concentration in BMA compared to PB. There was a 2.6-fold increase in monocyte concentration in BMA compared to PB. There was a 2.1-fold increase in lymphocyte concentration in BMA compared to PB. There was a 1.3-fold increase in platelet concentration in PB compared to BMA.
Flow Cytometry
Data regarding flow cytometry are illustrated in Figures 1 and 2. The percentage of T cells that expressed CD3+ across the 3 samples were as follows: PB 62.9% ± 13.8%, BMA 54.1% ± 14.6% and cBMA 59.3% ± 13.4%. There was a statistically significant higher percentage of T cells that were CD3+ in the PB compared to BMA (P = .0018).
Figure 1.
Cell surface markers. CD = cluster of differentiation.
Figure 2.
Flow cytometry. (A) White cell population. (B) CD3+ T Cells. (C) Classical CD14+ monocytes, Intermediate CD14+CD16+ monocytes and nonclassical CD16+ monocytes. (D) CD86+ M1 macrophages. (E). CD163+CD206+ M2 macrophages. CD = cluster of differentiation.
The percentage of monocytes that expressed CD14+ across the 3 samples were as follows: PB 73.3% ± 13.9%, BMA 78.5% ± 15.5%, and cBMA 75.2% ± 20.9%. The percentage of monocytes that were CD14+CD16+ across the 3 samples were as follows: PB 12.5% ± 7.4%, BMA 7.7% ± 6.1%, and cBMA 10.3% ± 8.0%. There was a statistically significant higher percentage of monocytes that expressed CD14+CD16+ in the PB compared to BMA (P = .0006). The percentage of monocytes that expressed CD16+ across the 3 samples were as follows: PB 14.6% ± 12.6%, BMA 13.1% ± 14.2%, and cBMA 14.9% ± 17.1%.
The percentage of monocytes that expressed CD86+ across the 3 samples were as follows: PB 90.5% ± 7.7%, BMA 90.2% ± 7.8% and cBMA 88.9% ± 8.8%. The percentage of monocytes that expressed CD163+CD206+ across the 3 samples were as follows: PB 7.9% ± 3.4%, BMA 5.7% ± 2.7%, and cBMA 5.6% ± 2.8%. There was a statistically significant higher percentage of monocytes that were CD163+CD206+ in the PB samples compared to BMA (P = .0004). There was a statistically significant higher percentage of monocytes that were CD163+CD206+ in the PB samples compared to BMA (P = .0002). The mean M1/M2 ratio for PB, BMA, and cBMA was 15.2 ± 12.0, 20.8 ± 13.3, and 22.1 ± 16.0, respectively ( Table 4 ).
Table 4.
M1/M2 Ratio, IL-1β, IL-1Ra, and IL-1Ra/IL-1β Ratio.
| PB | BMA | cBMA | |
|---|---|---|---|
| M1/M2 ratio | 15.2 ± 12.0 | 20.8 ± 13.3 | 22.1 ± 16.0 |
| IL-1β (pg/mL) | 2.45 ± 0.9 | 6.7 ± 7.6 | 31.4 ± 24.7 |
| IL-1Ra (pg/mL) | 1847.5 ± 1520.4 | 3143.0 ± 2218.5 | 8243.3 ± 14837.4 |
| IL-1Ra/IL-1β ratio | 790.6 ± 581.9 | 764.7 ± 675.2 | 235.7 ± 192.1 |
PB = peripheral blood; BMA = bone marrow aspirate; cBMA = concentrated bone marrow aspirate; WBC = white blood cell; IL-1β = interleukin-1β; IL-1Ra = interleukin-1 receptor antagonist.
IL-1β and IL-1Ra
Owing to technical processing difficulties, the multiplex assay was ran for only 36 samples of PB, 35 samples of BMA, and 30 samples of cBMA. All samples contained measurable IL-1β. There was a statistically significant higher concentration of Il-1β in the cBMA sample (31.4 ± 25.1 pg/mL) compared to both BMA (6.7 ± 7.8 pg/mL) and PB (2.5 ± 0.9 pg/mL) samples ( Table 4 ).
All samples contained measurable IL-1Ra. There was a statistically significant higher concentration of IL-1Ra in the cBMA sample (8243.3 ± 14,837.4 pg/mL) compared to both BMA (3143.0 ± 2218.5 pg/mL) and PB (1847.5 ± 1520.4 pg/mL).
The IL-1Ra/IL-1β ratio for PB, BMA, and cBMA was 790.6 ± 581.9, 764.7 ± 675.2, and 235.7 ± 192.1, respectively.
Comparison Between Centrifugation Systems
Data regarding the composition of cBMA between the Magellan (Isto Biologics, Hopkinton, Massachusetts) and Angel (Arthrex Inc, Naples, Florida) systems is listed in Table 5 . The Magellan system (Isto Biologics, Hopkinton, Massachusetts) produced a statistically significant higher fold change of monocytes (9.7 ± 9.1 vs 6.5 ± 8.4), platelets (14.3 ± 13.3 vs 8.6 ± 6.1), lymphocytes (11.6 ± 5.2 vs 6.9 ± 5.0), IL-1β concentrations (48.5 ± 31.1 pg/mL vs 22.8 ± 16.6 pg/mL), IL-1Ra concentrations (19,272.42 ± 25,779.1 pg/mL vs 4,232.7 ± 4,292 pg/mL), a higher percentage of classical CD14+ monocytes (84.8% ± 6.8% vs 69.7% ± 24.2%), and a higher percentage of intermediate CD14+CD16+ Monocytes (6.4% ± 3.5% vs 12.6% ± 9.1%) compared to the Angel system (Arthrex Inc, Naples, Florida).
Table 5.
Comparison of the Composition of cBMA between the Magellan and Arthrex Systems.
| Magellan | Arthrex | Significance | |
|---|---|---|---|
| WBC | 5.9 ± 2.7 | 3.9 ± 2.6 | P = .0873 |
| Neutrophil | 3.3 ± 2.4 | 2.4 ± 1.7 | P = .1880 |
| Monocyte | 9.7 ± 9.1 | 6.5 ± 8.4 | P = .0223 |
| Lymphocyte | 11.6 ± 5.2 | 6.9 ± 5.0 | P = .0223 |
| Platelet | 14.3 ± 13.4 | 8.6 ± 6.1 | P = .0355 |
| CD3+ T cell | 58.3 ± 7.3 | 59.8 ± 16.0 | P = .3559 |
| CD14+ monocytes | 84.8 ± 6.8 | 69.7 ± 24.2 | P = .0411 |
| CD14+CD16+ monocytes | 6.4 ± 3.5 | 12.6 ± 9.1 | P = .0251 |
| CD16+ monocytes | 8.4 ± 5.8 | 18.8 ± 20.3 | P = .2147 |
| CD86+ monocytes | 89.6 ± 6.7 | 88.6 ± 9.9 | P = .9517 |
| CD163+CD206+ monocytes | 5.9 ± 2.7 | 5.4 ± 2.9 | P = .5153 |
| M1/M2 ratio | 18.9 ± 11.1 | 24.0 ± 18.3 | P = .3045 |
| IL1-β | 48.5 ± 31.1 | 22.8 ± 16.6 | P = .0209 |
| IL1-Ra | 19,272.42 ± 25,779.1 | 4,232.7 ± 4,292 | P = .018 |
| Il1-Ra/IL-1β ratio | 1154.4 ± 2452.6 | 249.9 ± 186.7 | P = .5327 |
WBC = white blood cell; CD = clusters of differentiation; IL-1β = interleukin-1β; IL-1Ra = interleukin-1 receptor antagonist.
Bold values are statistically significant (p < 0.05)
There was no statistically significant difference in the fold change of WBCs (5.9 ± 2.7 vs 3.9 ± 2.6), neutrophils (3.2 ± 2.4 vs 2.4 ± 1.7), Il-1Ra/IL-1β ratio (1154.4 ± 2452.6 vs 249.9 ± 186.7), or M1/M2 ratio (19.0 ± 11.1 vs 24.0 ± 18.3) between the Magellan (Isto Biologics, Hopkinton, Massachusetts) and Angel systems (Arthrex Inc, Naples, Florida).
Discussion
The most important finding of this study was that the M1/M2 ratio in cBMA was 22.1 ± 16.0, with significant patient to patient variation observed. Overall, there was no statistically significant difference in the M1/M2 ratio across PB, BMA, and cBMA samples. This is the first study to characterize the macrophage subpopulation within cBMA, which may have significant clinical implications in future studies.
Injury to the articular cartilage triggers a cascade of events involving various inflammatory cells and mediators within the joint microenvironment, producing both degradative and reparative responses. 8 The balance between inflammatory and reparative processes dictates the outcome of cartilage injury, with prolonged inflammation disrupting the delicate equilibrium necessary for effective repair. 8 Monocytes and macrophages infiltrate synovial tissue upon cartilage damage, secreting inflammatory mediators in order to clear damaged tissues. 9 Macrophages exhibit a high degree of plasticity, with M1 macrophages activated early via toll-like receptors and LPS, promoting inflammation and cartilage destruction. 10 Conversely, M2 macrophages, induced by Th2 cell-secreted IL-4 and IL-13, produce anti-inflammatory cytokines and support chondrogenesis, aiding in cartilage repair.8,10 M1 macrophages hinder MSC differentiation and exacerbate extracellular matrix degradation, whereas M2 macrophages promote tissue regeneration by secreting growth factors and matrix components. 8 Overall, M2 macrophages play a crucial role in cartilage repair, counteracting the detrimental effects of M1 macrophages. 7 Understanding the balance between M1 and M2 macrophages is essential for developing targeted therapies for cartilage repair, and manipulating the polarization of macrophages toward the M2 phenotype may offer promising strategies for enhancing cartilage repair and mitigating inflammation-induced tissue damage.
This study found that there was significant variation in the M1/M2 ratio in cBMA from patient to patient, ranging from 7 to 80. No patients in this cohort had a history of disorders that are known to affect the M1/M2 ratio such as inflammatory arthritis, diabetes, and cancer. For patients with a more favorable M1/M2 ratio in cBMA, it is plausible that delivery of a predominant anti-inflammatory macrophage population to the injured cartilage may improve the biological mileu, thus propagating superior cartilage tissue repair compared to patients receiving cBMA containing a higher, more pro-inflammatory M1/M2 macrophage population. Subsequent longitudinal studies are necessary to ascertain the correlation between the patient-specific M1/M2 ratio contained within cBMA, and the clinical and radiological outcomes following the use of cBMA for the treatment of OLTs.
This study sought to determine the M1/M2 ratio not only in cBMA but in corresponding PB and BMA samples. There is discordance in the literature regarding the normal M1/M2 ratio in PB in healthy controls compared to patients with arthritic conditions. It is commonly stated in the literature that an M1/M2 ratio greater than 1 is consistent with a pro-inflammatory milieu 11 ; however, there is no definitive evidence to support this suggestion. Fukui et al evaluated the M1/M2 ratio in patients with rheumatoid arthritis (RA) and healthy controls and determined the mean M1/M2 ratio in the RA cohort to be 0.60 and the mean M1/M2 ratio in the healthy control cohort to be 0.62. 11 Furthermore, Liu et al found that the mean M1/M2 ratio in healthy controls to be approximately 1.1 with the mean M1/M2 ratio in patients with knee osteoarthritis approximately 4.3. 12 The mean M1/M2 ratio in PB in this study was 15.2 ± 12.0, markedly higher than previous studies, which may be attributed to different flow cytometry protocols implemented between studies. There is a paucity of data regarding the M1/M2 ratio in BMA. Zhang et al compared the M1/M2 ratio in patients with myelodysplastic syndrome to healthy controls and found that the mean M1/M2 ratio in the MDS cohort was 1.80 ± 0.9 compared to 3.5 ± 3.2 in the healthy control cohort. 13 Similar to PB, there was a significantly higher M1/M2 ratio in the BMA samples (20.8 ± 13.3) in this study compared to prior studies.
Although the M1/M2 ratio may play a key role in the anti-inflammatory properties exerted by cBMA, the involvement of other essential anti-inflammatory factors must be examined. IL-1, initially identified in the 1940s as a pyrexia-inducing molecule, is a powerful pro-inflammatory cytokine. 14 IL-1 exists in 2 main isoforms, IL-1α and IL-1β, both synthesized as precursor peptides that require cleavage to become active. 15 IL-1β binds to its receptor, IL-1RI, initiating downstream signaling cascades that lead to the recruitment of inflammatory cells, the production of pro-inflammatory mediators and the inhibition of proteoglycan and type II collagen production, promoting the destruction of the articular cartilage. 15 In addition, IL-1β stimulates the maturation of osteoclasts, contributing to bony erosions. IL-1Ra, a naturally occurring cytokine, acts as a specific receptor antagonist for IL-1. IL-1Ra is produced in multiple isoforms, including secreted and intracellular forms, each contributing to the modulation of IL-1 signaling. 16 The secreted form of IL-1Ra (sIL-1Ra) acts extracellularly to inhibit IL-1 activity, while intracellular isoforms can also exert inhibitory effects on IL-1 signaling by binding to IL-1RI intracellularly, thereby preventing receptor activation. 16 This study demonstrated that cBMA contains abundant levels of IL-1Ra (8243.3 ± 14837.4 pg/mL), at a significantly higher concentration compared to both BMA (3143.0 ± 2218.5 pg/mL) and PB (1847.5 ± 1520.4 pg/mL). In addition, this study found that cBMA contains a favorable IL-1Ra: IL-1β ratio (235.7 ± 192.0), surpassing the minimum threshold warranted to effectively inhibit IL-1β (>10:1 to 100:1). This finding is consistent with previous studies, which also demonstrated a high IL-1Ra: IL-1β ratio contained within cBMA. 5
This study sought to compare the composition of cBMA generated by 2 commercially available BMA concentration systems. The Angel (Arthrex Inc, Naples, Florida) and Magellan (Isto Biologics, Hopkinton, Massachusetts) systems exhibit similar product characteristics and specifications. Both are fully automated, computer-controlled, closed-loop systems equipped with an integrated centrifuge. These devices incorporate an optical sensor to detect various blood fractions, facilitating the separation of the buffy coat postcentrifugation. In a fully automated setup, users simply connect the BMA syringe, input parameters via computer software, and the system autonomously delivers the BMC in a ready-to-use syringe, eliminating the need for manual switching or additional user interventions. 17 Advantages of an integrated centrifuge includes procedural room efficiency and reduced equipment requirements. 17 While the benefits of a closed-loop system include enhanced convenience and potentially reduced infection or contamination risks, there is scant evidence to support these proposed benefits. 17 Overall, there were some differences in cBMA composition between the 2 systems. The Magellan system (Isto Biologics, Hopkinton, Massachusetts) produced a higher concentration of monocytes, platelets, lymphocytes, classical CD14+ monocytes and intermediate CD14+CD16+ monocytes compared to the Angel (Arthrex Inc, Naples, Florida) system, which may be attributed to the differences in centrifugation protocols. Theoretically, delivery of a higher concentration of these immune cells, platelets and IL-1Ra may alter the biology of the local microenvironment within the joint, thus potentially producing difference in reparative tissue quality. However, there was no difference in the M1/M2 ratio nor IL-1Ra/IL1-β ratio between the 2 systems, thus it cannot be determined at present if the differences in cellular composition between the 2 systems will have any substantial impact on cartilage repair.
This study must be interpreted in light of its numerous limitations and inherent biases. First, no data regarding clinical and radiological outcomes were obtained, so it is currently unknown if the M1/M2 macrophage ratio in cBMA will have any appreciable effects on these outcomes. Furthermore, the sample size was small, and no adequate control cohort of healthy patients was utilized in this study. In addition, certain medical co-morbidities that have been demonstrated to affect macrophage, and monocyte counts were not included in the exclusion criteria such as cardiovascular disease, coronary artery disease, LV dysfunction, LV aneurysm, chronic kidney disease, heart failure, or a history of stroke.
Conclusion
This prospective study found that the M1/M2 ratio in cBMA was 22.1 ± 16.0, with significant patient to patient variation observed. Overall, there was no statistically significant difference in the M1/M2 ratio across PB, BMA, and cBMA samples. This is the first study to characterize the macrophage subpopulation within cBMA, which may have significant clinical implications in future studies.
Footnotes
Acknowledgments and Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article.
The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: J.G.K. is a consultant for Arteriocyte, In2Bones, and Arthrex. J.G.K. receives financial support from the Ohnell Family Foundation, Mr Winston Fischer and Tatiana Rybak.
Ethical Approval Statement: Ethical approval was obtained following Institutional Review Board approval (i21-00163)
ORCID iDs: James J. Butler 
https://orcid.org/0000-0002-4212-5018
John F. Dankert
https://orcid.org/0000-0003-1443-9527
Jari Dahmen
https://orcid.org/0000-0002-6849-1008
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