The Choice of Anticoagulant Influences the Characteristics of Bone Marrow Aspirate Concentrate and Mesenchymal Stem Cell Bioactivity In Vitro.

Bone marrow aspirate concentrate (BMC) is commonly used as a therapeutic agent to resolve orthopedic injuries, using its unique cellularity to reduce inflammation and prime the region for repair. The aspiration of the bone marrow is performed using either sodium citrate (SC) or heparin sodium (HS) as an anticoagulant and processed via centrifugation to concentrate the cellular constituents. To date, the consideration of the impact of the two commonly used anticoagulants on the mesenchymal stem/stromal cell (MSC) population has been overlooked. The current study assesses the differences in the BMCs produced using 15% SC and HS at 1,000 U/mL or 100 U/mL final v./v. as an anticoagulant using in vitro metrics including total nucleated cell counts (TNC) and viability, the ability for mesenchymal stromal/stem cells (MSCs) to establish colony-forming units with fibroblast morphology (CFU-f), and cytokine expression profile of the MSC cultures. Our findings demonstrate that HS-derived BMC cultures result in higher CFU-f formation and CFU-f frequency at both concentrations assessed compared to SC-derived BMC cultures. In addition, there were significant differences in 27% (7 of 26) of the cytokines quantified in HS-derived BMC cultures compared to SC-derived BMC cultures with implications for MSC plasticity and self-renewal.

Soluble factors differ in platelets derived from separate niches: a pilot study comparing the secretome of peripheral blood and bone marrow platelets.

BACKGROUND AIMS: Platelet-rich plasma (PRP) and bone marrow aspirate are commonly used in orthobiologics for their anti-inflammatory, anabolic/regenerative and immunomodulatory characteristics via platelet degranulation and cell secretions. Although platelets are derived from megakaryocytes in the bone marrow, no attention has been paid to the potential benefits of bone marrow platelets and whether their contents differ from aging platelets in peripheral blood. METHODS: In the present study, leukocyte-poor peripheral blood-derived platelets in plasma (LPP) and leukocyte-poor bone marrow platelets in plasma (BMP) were prepared from six donors, activated with calcium chloride, incubated and sampled at day 0, day 3 and day 6. LPP and BMP are platelet preparations intended to evaluate the respective platelet secretomes but are not classified as conventional PRPs, as they are not concentrated to the extent necessary to meet the qualifying criteria. At each time point, 15 growth and immunomodulatory factors were quantitated in LPP and BMP: platelet-derived growth factor AA, basic fibroblast growth factor/fibroblast growth factor 2, granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, macrophage colony-stimulating factor, stem cell factor, vascular endothelial growth factor, tumor necrosis factor alpha, IL-1ß, interferon gamma, IL-4, IL-10, IL-1 receptor antagonist protein, IL-12p40 and arginase-1. RESULTS: The results illustrate that platelets derived from bone marrow have a unique secretome profile compared with those derived from peripheral blood, with significant differences in anti-inflammatory cytokines, which are associated with monocyte polarization. CONCLUSIONS: Ultimately, bone marrow-derived platelets may be useful as a stand-alone orthobiologic or as an effective adjuvant to autologous cell therapies where anti-inflammatory and anabolic processes are desired, especially with respect to monocyte function.

Effect of local anesthetics on platelet physiology and function.

Local anesthetics are often used at the site of injury or mixed with platelet-rich plasma to reduce pain when treating orthopedic and sports-related injuries. Local anesthetics have been shown to have deleterious effects on stromal cells, but their impact on platelets has not been investigated. In this study, we aimed to assess the effects of lidocaine, bupivacaine, and ropivacaine on platelet health. Based on the deleterious effects of local anesthetics on nucleated cells, we hypothesized that these compounds would affect platelet viability, intracellular physiology, and function. Platelet preparations were derived from randomly selected donors and exposed to lidocaine 1%, bupivacaine 0.75%, ropivacaine 0.5%, and saline at 1:1 and 1:3 ratios. Platelet morphology, viability, intracellular calcium, production of radical oxygen species (ROS), apoptosis, and adhesion were assessed via fluorescent microscopy and flow cytometry. Bupivacaine resulted in increased ROS production, calcium dysregulation, apoptosis, and reduced platelet adhesion. By contrast, ropivacaine and lidocaine were similar to saline in most assays, except for a low degree of mitochondrial stress as evidenced by increased ROS production. Ultimately, bupivacaine 0.75% was harmful to platelets as evidenced by reduced platelet viability, adhesion, and increased apoptosis, whereas lidocaine 1% and ropivacaine 0.5% were relatively safe at the 1:1 and 1:3 dilutions. Clinical significance: Lidocaine 1% and ropivacaine 0.5% can be used at up to a 1:1 ratio with platelet preparations to reduce the pain and discomfort of PRP procedures while maintaining platelet therapeutic potential.

Human mesenchymal stem cells respond differentially to platelet preparations and synthesize hyaluronic acid in nucleus pulposus extracellular matrix.

BACKGROUND CONTEXT: In recent years, autologous platelet-rich plasma (PRP) and bone marrow aspirate concentrate (BMAC) have been used as treatments for disc-related pain. A better understanding of the effects of leukocyte-rich (LR) versus leukocyte poor (LP-) PRP on bone marrow derived human mesenchymal stem/progenitor cells (hMSCs) is likely to improve future research studies, clinical practice and care for patients with chronic discogenic back pain. PURPOSE: The primary aim of this study is to determine the effects of LR-PRP and LP-PRP on the proliferation and migration of hMSCs in pig nucleus pulposus (NP) extracellular matrix (ECM). The secondary aim is to characterize hMSC-dependent expression of the matrix remodeling enzymes metalloproteinases MMP-2, MMP-3, MMP-9 and tissue inhibitor of metalloproteinases TIMP-2, and to determine whether transplanted hMSCs can synthesize hyaluronic acid (HA). STUDY DESIGN: Controlled laboratory study. METHODS: Bone marrow-derived culture expanded hMSCs were seeded onto pig NP and cultured with LR-PRP, LP-PRP or serum/platelet releasate (PR). The same conditions without hMSCs were used as controls. hMSC proliferation, migration and dispersion was assessed via fluorescent microscopy, while HA synthesis, MMP-2, MMP-3, MMP-9, and TIMP-2 protein levels were assessed via enzyme linked immunosorbent assay. All funding was provided by a 501c(3) research foundation and does not have any commercial or sponsorship interests. RESULTS: LP-PRP and PR cultures resulted in higher hMSC proliferation, migration, dispersion, and MMP-2 expression. LP-PRP cultures resulted in the highest HA production. LR-PRP cultures resulted in lower hMSC proliferation, negligible migration and dispersion, increased MMP-9 expression and lower HA production. CONCLUSIONS: Human bone marrow-derived hMSCs seeded onto pig NP ECM are capable of synthesizing HA, indicating a transition towards a NP cell phenotype. This process was most enhanced by LP-PRP and marked by increased hMSC proliferation, MMP-2 production, HA synthesis and reduced MMP-9 levels. CLINICAL SIGNIFICANCE: LP-PRP and PR, with or without hMSCs, may provide better outcomes than LR-PRP in lab investigations and clinical trials for discogenic pain. Bone marrow-derived hMSCs may hold promise as a treatment for disc degeneration.

A fibrin/hyaluronic acid hydrogel for the delivery of mesenchymal stem cells and potential for articular cartilage repair.

BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease affecting approximately 27 million Americans, and even more worldwide. OA is characterized by degeneration of subchondral bone and articular cartilage. In this study, a chondrogenic fibrin/hyaluronic acid (HA)-based hydrogel seeded with bone marrow-derived mesenchymal stem cells (BMSCs) was investigated as a method of regenerating these tissues for OA therapy. This chondrogenic hydrogel system can be delivered in a minimally invasive manner through a small gauge needle, forming a three-dimensional (3D) network structure in situ. However, an ongoing problem with fibrin/HA-based biomaterials is poor mechanical strength. This was addressed by modifying HA with methacrylic anhydride (MA) (HA-MA), which reinforces the fibrin gel, thereby improving mechanical properties. In this study, a range of fibrinogen (the fibrin precursor) and HA-MA concentrations were explored to determine optimal conditions for increased mechanical strength, BMSC proliferation, and chondrogenesis potential in vitro. RESULTS: Increased mechanical strength was achieved by HA-MA reinforcement within fibrin hydrogels, and was directly correlated with increasing HA-MA concentration. Live/dead staining and metabolic assays confirmed that the crosslinked fibrin/HA-MA hydrogels provided a suitable 3D environment for BMSC proliferation. Quantitative polymerase chain reaction (qPCR) of BMSCs incubated in the fibrin/HA-MA hydrogel confirmed decreased expression of collagen type 1 alpha 1 mRNA with an increase in Sox9 mRNA expression especially in the presence of a platelet lysate, suggesting early chondrogenesis. CONCLUSION: Fibrin/HA-MA hydrogel may be a suitable delivery method for BMSCs, inducing BMSC differentiation into chondrocytes and potentially aiding in articular cartilage repair for OA therapy.

Amide-type local anesthetics and human mesenchymal stem cells: clinical implications for stem cell therapy.

In the realm of regenerative medicine, human mesenchymal stem cells (hMSCs) are gaining attention as a cell source for the repair and regeneration of tissues spanning an array of medical disciplines. In orthopedics, hMSCs are often delivered in a site-specific manner at the area of interest and may require the concurrent application of local anesthetics (LAs). To address the implications of using hMSCs in combination with anesthetics for intra-articular applications, we investigated the effect that clinically relevant doses of amide-type LAs have on the viability of bone marrow-derived hMSCs and began to characterize the mechanism of LA-induced hMSC death. In our study, culture-expanded hMSCs from three donors were exposed to the amide-type LAs ropivacaine, lidocaine, bupivacaine, and mepivacaine. To replicate the physiological dilution of LAs once injected into the synovial capsule, each anesthetic was reduced to 12.5%, 25%, and 50% of the stock solution and incubated with each hMSC line for 40 minutes, 120 minutes, 360 minutes, and 24 hours. At each time point, cell viability assays were performed. We found that extended treatment with LAs for 24 hours had a significant impact on both hMSC viability and adhesion. In addition, hMSC treatment with three of the four anesthetics resulted in cell death via apoptosis following brief exposures. Ultimately, we concluded that amide-type LAs induce hMSC apoptosis in a time- and dose-dependent manner that may threaten clinical outcomes, following a similar trend that has been established between these particular anesthetics and articular chondrocytes both in vitro and in vivo.

Erratum to: A fibrin/hyaluronic acid hydrogel for the delivery of mesenchymal stem cells and potential for articular cartilage repair.

[This corrects the article DOI: 10.1186/1754-1611-8-10.].

Murine Prkdc polymorphisms impact DNA-PKcs function.

Polymorphic variants of DNA repair genes can increase the carcinogenic potential of exposure to ionizing radiation. Two single nucleotide polymorphisms (SNPs) in Prkdc, the gene encoding the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), have been identified in BALB/c mice and linked to reduced DNA-PKcs activity and mammary cancer susceptibility. We examined three additional mouse strains to better define the roles of the BALB/c Prkdc SNPs (R2140C and M3844V). One is a congenic strain (C.B6) that has the C57BL/6 Prkdc allele on a BALB/c background, and the other is a congenic strain (B6.C) that has the BALB/c variant Prkdc allele on a C57BL/6 background. We also examined the LEWES mouse strain, which possesses only one of the BALB/c Prkdc SNPs (M3844V). Our results demonstrate that both Prkdc SNPs are responsible for deficient DNA-PKcs protein expression, DNA repair and telomere function, while the LEWES SNP affects only DNA-PKcs expression and repair capacity. These studies provide insight into the separation of function between the two BALB/c SNPs as well as direct evidence that SNPs positioned within Prkdc can significantly influence DNA-PKcs function involving DNA repair capacity, telomere end-capping, and potentially cancer susceptibility.

Regulatory roles of tankyrase 1 at telomeres and in DNA repair: suppression of T-SCE and stabilization of DNA-PKcs.

Intrigued by the dynamics of the seemingly contradictory yet integrated cellular responses to the requisites of preserving telomere integrity while also efficiently repairing damaged DNA, we investigated roles of the telomere associated poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) tankyrase 1 in both telomere function and the DNA damage response following exposure to ionizing radiation. Tankyrase 1 siRNA knockdown in human cells significantly elevated recombination specifically within telomeres, a phenotype with the potential of accelerating cellular senescence. Additionally, depletion of tankyrase 1 resulted in concomitant and rapid reduction of the nonhomologous end-joining protein DNA-PKcs, while Ku86 and ATM protein levels remained unchanged; DNA-PKcs mRNA levels were also unaffected. We found that the requirement of tankyrase 1 for DNA-PKcs protein stability reflects the necessity of its PARP enzymatic activity. We also demonstrated that depletion of tankyrase 1 resulted in proteasome-mediated DNA-PKcs degradation, explaining the associated defective damage response observed; i.e., increased sensitivity to ionizing radiation-induced cell killing, mutagenesis, chromosome aberration and telomere fusion. We provide the first evidence for regulation of DNA-PKcs by tankyrase 1 PARP activity and taken together, identify roles of tankyrase 1 with implications not only for DNA repair and telomere biology, but also for cancer and aging.