How to obtain and isolate equine sternal bone marrow mononuclear cells with limited resources

Correa, Francisco; Borlone, C; Wittwer, F; Bustamante, Hedie; Müller, Albrecht; Ramírez, A; Menarim, Bruno Carvalho

doi:10.4067/s0301-732x2014000300019

Cited by 4 publications

(9 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Horses were sedated and the sternum clipped, aseptically prepared, and locally anesthetized. Heparinized bone marrow aspirate was collected from the fourth and fifth sternebrae using an 11 gauge Jamshidi needle . In a laminar flow hood, bone marrow aspirate was filtered, gently layered over density gradient medium (Ficoll‐Paque™ Plus; GE Healthcare Life Sciences, Pittsburgh, PA), and centrifuged at 500 g for 30 min at 4°C .…”

Section: Methodsmentioning

confidence: 99%

“…Heparinized bone marrow aspirate was collected from the fourth and fifth sternebrae using an 11 gauge Jamshidi needle . In a laminar flow hood, bone marrow aspirate was filtered, gently layered over density gradient medium (Ficoll‐Paque™ Plus; GE Healthcare Life Sciences, Pittsburgh, PA), and centrifuged at 500 g for 30 min at 4°C . After supernatant removal, the mononuclear cells in Ficoll were aspirated, transferred to a sterile centrifuge tube, washed in Dulbecco's phosphate‐buffered saline (DPBS), centrifuged at 300 g for 10 min at 4°C, the cell pellet resuspended in growth medium (Mesenchymal Stem Cell Medium with 5% fetal bovine serum [FBS]; ScienCell Research Laboratories, Carlsbad, CA) at 37°C, 5% CO 2 , and 95% humidity, and passaged routinely using trypsin/ethylenediaminetetraacetic acid prior to cells reaching 70% confluence.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Urinary Bladder Matrix Does Not Improve Tenogenesis in an In Vitro Equine Model

Khatibzadeh

Menarim

Nichols

et al. 2019

Journal Orthopaedic Research

View full text Add to dashboard Cite

Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels seeded with bone marrow cells (n = 6 adult horses) were cast using rat tail tendon ECM ± UBM, fixed under static tension and harvested at 7 and 21 days for construct contraction, cell viability, histology, biochemistry, and gene expression. By day 7, UBM constructs contracted significantly from baseline, whereas control constructs did not. Both control and UBM constructs contracted significantly by day 21. In both groups, cells remained viable over time and changed from round and randomly oriented to elongated along lines of tension with visible compaction of the ECM. There were no differences over time or between treatments for nuclear aspect ratio, DNA, or glycosaminoglycan content. Decorin, matrix metalloproteinase 13, and scleraxis expression increased significantly over time, but not in response to UBM treatment. Mohawk expression was constant over time. Cartilage oligomeric matrix protein expression decreased over time in both groups. Using a novel ECM hydrogel model, substantial matrix organization and cell differentiation occurred; however, the addition of UBM failed to induce greater matrix organization than tendon ECM alone.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Urinary Bladder Matrix Does Not Improve Tenogenesis in an In Vitro Equine Model

Khatibzadeh

Menarim

Nichols

et al. 2019

Journal Orthopaedic Research

View full text Add to dashboard Cite

show abstract

“…BMNC isolation from bone marrow aspirates using gradient centrifugation is rapid and can be completed with minimal manipulation. Cells are readily available for immediate administration using few equipment[ 90 , 95 , 96 ]. The resulting BMNC cell isolate is composed primarily of mononuclear cells (> 90%).…”

Section: Bone Marrow Mononuclear Cells As a Therapeutic Source Of Healthy Macrophagesmentioning

confidence: 99%

“…The result is a relative dilution of the desired BMNC with peripheral cells. One useful parameter in assessing if the bone marrow aspirate volume is excessive and diluted by peripheral blood is the platelet count, given that the concentration of platelets in the bone marrow is very low[ 96 ]. A detailed protocol for harvesting equine bone marrow aspirates and processing BMNC with minimal resources have been described elsewhere[ 96 ].…”

Section: Bone Marrow Mononuclear Cells As a Therapeutic Source Of Healthy Macrophagesmentioning

confidence: 99%

“…One useful parameter in assessing if the bone marrow aspirate volume is excessive and diluted by peripheral blood is the platelet count, given that the concentration of platelets in the bone marrow is very low[ 96 ]. A detailed protocol for harvesting equine bone marrow aspirates and processing BMNC with minimal resources have been described elsewhere[ 96 ]. While only 20-40 x 10 6 BMNC are used for joint injection, a bone marrow aspirate of 25 mL yields 150-200 x 10 6 BMNC and the exceeding cells can be cryopreserved for future treatments without compromising BMNC function[ 127 ].…”

Section: Bone Marrow Mononuclear Cells As a Therapeutic Source Of Healthy Macrophagesmentioning

confidence: 99%

See 1 more Smart Citation

Bone marrow mononuclear cells for joint therapy: The role of macrophages in inflammation resolution and tissue repair

Menarim¹,

MacLeod²,

Dahlgren³

2021

WJSC

View full text Add to dashboard Cite

Osteoarthritis (OA) is the most prevalent joint disease causing major disability and medical expenditures. Synovitis is a central feature of OA and is primarily driven by macrophages. Synovial macrophages not only drive inflammation but also its resolution, through a coordinated, simultaneous expression of pro- and anti-inflammatory mechanisms that are essential to counteract damage and recover homeostasis. Current OA therapies are largely based on anti-inflammatory principles and therefore block pro-inflammatory mechanisms such as prostaglandin E 2 and Nuclear factor-kappa B signaling pathways. However, such mechanisms are also innately required for mounting a pro-resolving response, and their blockage often results in chronic low-grade inflammation. Following minor injury, macrophages shield the damaged area and drive tissue repair. If the damage is more extensive, macrophages incite inflammation recruiting more macrophages from the bone marrow to maximize tissue repair and ultimately resolve inflammation. However, sustained damage and inflammation often overwhelms pro-resolving mechanisms of synovial macrophages leading to the chronic inflammation and related tissue degeneration observed in OA. Recently, experimental and clinical studies have shown that joint injection with autologous bone marrow mononuclear cells replenishes inflamed joints with macrophage and hematopoietic progenitors, enhancing mechanisms of inflammation resolution, providing remarkable and long-lasting effects. Besides creating an ideal environment for resolution with high concentrations of interleukin-10 and anabolic growth factors, macrophage progenitors also have a direct role in tissue repair. Macrophages constitute a large part of the early granulation tissue, and further transdifferentiate from myeloid into a mesenchymal phenotype. These cells, characterized as fibrocytes, are essential for repairing osteochondral defects. Ongoing “omics” studies focused on identifying key drivers of macrophage-mediated resolution of joint inflammation and those required for efficient osteochondral repair, have the potential to uncover ways for developing engineered macrophages or off-the-shelf pro-resolving therapies that can benefit patients suffering from many types of arthropaties, not only OA.

show abstract

Equine bone marrow-derived mesenchymal stromal cells reduce established S. aureus and E. coli biofilm matrix in vitro

Khatibzadeh,

Dahlgren,

Caswell

et al. 2024

PLoS ONE

View full text Add to dashboard Cite

Biofilms reduce antibiotic efficacy and lead to complications and mortality in human and equine patients with orthopedic infections. Equine bone marrow-derived mesenchymal stromal cells (MSC) kill planktonic bacteria and prevent biofilm formation, but their ability to disrupt established orthopedic biofilms is unknown. Our objective was to evaluate the ability of MSC to reduce established S. aureus or E. coli biofilms in vitro. We hypothesized that MSC would reduce biofilm matrix and colony-forming units (CFU) compared to no treatment and that MSC combined with the antibiotic, amikacin sulfate, would reduce these components more than MSC or amikacin alone. MSC were isolated from 5 adult Thoroughbred horses in antibiotic-free medium. 24-hour S. aureus or E. coli biofilms were co-cultured in triplicate for 24 or 48 hours in a transwell plate system: untreated (negative) control, 30 μg/mL amikacin, 1 x 106 passage 3 MSC, and MSC with 30 μg/mL amikacin. Treated biofilms were photographed and biofilm area quantified digitally. Biomass was quantified via crystal violet staining, and CFU quantified following enzymatic digestion. Data were analyzed using mixed model ANOVA with Tukey post-hoc comparisons (p < 0.05). MSC significantly reduced S. aureus biofilms at both timepoints and E. coli biofilm area at 48 hours compared to untreated controls. MSC with amikacin significantly reduced S. aureus biofilms versus amikacin and E. coli biofilms versus MSC at 48 hours. MSC significantly reduced S. aureus biomass at both timepoints and reduced S. aureus CFU at 48 hours versus untreated controls. MSC with amikacin significantly reduced S. aureus biomass versus amikacin at 24 hours and S. aureus and E. coli CFU versus MSC at both timepoints. MSC primarily disrupted the biofilm matrix but performed differently on S. aureus versus E. coli. Evaluation of biofilm-MSC interactions, MSC dose, and treatment time are warranted prior to testing in vivo.

show abstract

How to obtain and isolate equine sternal bone marrow mononuclear cells with limited resources

Cited by 4 publications

References 17 publications

Urinary Bladder Matrix Does Not Improve Tenogenesis in an In Vitro Equine Model

Urinary Bladder Matrix Does Not Improve Tenogenesis in an In Vitro Equine Model

Bone marrow mononuclear cells for joint therapy: The role of macrophages in inflammation resolution and tissue repair

Equine bone marrow-derived mesenchymal stromal cells reduce established S. aureus and E. coli biofilm matrix in vitro

Contact Info

Product

Resources

About