Isolation and characterisation of nasoseptal cartilage stem/progenitor cells and their role in the chondrogenic niche.

BACKGROUND: Since cartilage-derived stem/progenitor cells (CSPCs) were first identified in articular cartilage using differential adhesion to fibronectin, their self-renewal capacity and niche-specific lineage preference for chondrogenesis have propelled their application for cartilage tissue engineering. In many adult tissues, stem/progenitor cells are recognised to be involved in tissue homeostasis. However, the role of nasoseptal CSPCs has not yet been elucidated. Our aim was to isolate and characterise nasoseptal CSPCs alongside nasoseptal chondrocyte populations and determine chondrogenic capacity. METHODS: Here, we isolated nasoseptal CSPCs using differential adhesion to fibronectin and assessed their colony forming efficiency, proliferation kinetics, karyotype and trilineage potential. CSPCs were characterised alongside non-fibronectin-adherent nasoseptal chondrocytes (DNCs) and cartilage-derived cells (CDCs, a heterogenous combination of DNCs and CSPCs) by assessing differences in gene expression profiles using PCR Stem Cell Array, immunophenotype using flow cytometry and chondrogencity using RT-PCR and histology. RESULTS: CSPCs were clonogenic with increased gene expression of the neuroectodermal markers NCAM1 and N-Cadherin, as well as Cyclins D1 and D2, compared to DNCs. All three cell populations expressed recognised mesenchymal stem cell surface markers (CD29, CD44, CD73, CD90), yet only CSPCs and CDCs showed multilineage differentiation potential. CDC populations expressed significantly higher levels of type 2 collagen and bone morphogenetic protein 2 genes, with greater cartilage extracellular matrix secretion. When DNCs were cultured in isolation, there was reduced chondrogenicity and higher expression of type 1 collagen, stromal cell-derived factor 1 (SDF-1), CD73 and CD90, recognised markers of a fibroblast-like phenotype. CONCLUSIONS: Fibronectin-adherent CSPCs demonstrate a unique gene expression profile compared to non-fibronectin-adherent DNCs. DNCs cultured in isolation, without CSPCs, express fibroblastic phenotype with reduced chondrogenicity. Mixed populations of stem/progenitor cells and chondrocytes were required for optimal chondrogenesis, suggesting that CSPCs may be required to retain phenotypic stability and chondrogenic potential of DNCs. Crosstalk between DNCs and CSPCs is proposed based on SDF-1 signalling.

Virtual implantations to transition from porcine to bovine animal models for a total artificial heart.

Realheart total artificial heart (TAH) is a novel, pulsatile, four-chamber total artificial heart which had been successfully tested acutely in a porcine animal model. However, the bovine model is better suited for long-term testing and thus an evaluation of how the design would fit the bovine anatomy was required. Virtual implantation is a method that enables a computer simulated implantation based on anatomical 3D-models created from computer tomography images. This method is used clinically, but not yet adopted for animal studies. Herein, we evaluated its suitability in the redesign of the outer dimensions and vessel connections of Realheart TAH to transition from the porcine to the bovine animal model. Virtual implantations in combination with bovine cadaver studies enabled a series of successful acute bovine implantations. Virtual implantations are a useful tool to replace the use of animals in early device development and refine subsequent necessary in vivo experiments. The next steps are to carry out human virtual implantations and cadaver studies to ensure the design is optimized for all stages of testing as well as the final recipient.

In Vitro Benchmarking Study of Ventricular Assist Devices in Current Clinical Use.

BACKGROUND: Left ventricular assist devices (LVADs) offer live-saving therapy to transplant-ineligible heart failure patients. A major limitation of the technology includes pump thrombosis, bleeding, and recurrent infection that prove difficult to predict from in vivo animal testing. Shear stress introduced by the LVAD affects more than just hemolysis because platelets, leukocytes, and plasma proteins all contribute to the propensity for complications. It is important to assess overall damage by a new device against a baseline as early as possible in the development process so that design iterations can be made if required. METHODS: Explanted VADs currently in clinical use (HeartMate 2 and HVAD) were carefully cleaned, inspected, and run at 5 L/min and pressure at 100 mmHg in a standard 500 mL mock circulatory loop using bovine blood. The CentriMag was used as a control pump because of its low blood damage profile. Samples were collected at regular intervals and the following were analyzed: complete cell counts, hemolysis, platelet activation, leukocyte-derived microparticles (LMPs), and von Willebrand factor (vWF) degradation. RESULTS: The HeartMate 2 had the highest levels of hemolysis and platelet activation after 6 hours compared with the HVAD and CentriMag. A decreased granulocyte count, high numbers of LMPs and CD11bBrightHLADR- LMPs, and decreased vWF collagen binding activity was most evident in the HVAD. CONCLUSIONS: The results indicate that it is possible to observe differences between different pump designs during in vitro testing that might translate to clinical performance. This study demonstrates the importance of developing standard in vitro total blood damage methods against which device developers could use to modify design to reduce complication risk long before implantation.

Evaluation of the novel total artificial heart Realheart in a pilot human fitting study.

Heart failure affects >26 million patients worldwide. Current cardiac devices save lives, but patients suffer complications. Hence, improved devices are needed. Realheart TAH is a novel total artificial heart which has shown promising results in acute pig studies. However, the device design needed to be evaluated in humans. Virtual implantations demonstrated the device fits in two of three patients, but that there was some interference with the left lung. Herein, we used an innovative 3D-printed model with swivelling device components to test the device in human cadavers. Our new method demonstrated how to optimize design to improve the surgical fit.

Mechanical shear stress and leukocyte phenotype and function: Implications for ventricular assist device development and use.

Heart failure remains a disease of ever increasing prevalence in the modern world. Patients with end-stage heart failure are being referred increasingly for mechanical circulatory support. Mechanical circulatory support can assist patients who are ineligible for transplant and stabilise eligible patients prior to transplantation. It is also used during cardiopulmonary bypass surgery to maintain circulation while operating on the heart. While mechanical circulatory support can stabilise heart failure and improve quality of life, complications such as infection and thrombosis remain a common risk. Leukocytes can contribute to both of these complications. Contact with foreign surfaces and the introduction of artificial mechanical shear stress can lead to the activation of leukocytes, reduced functionality and the release of pro-inflammatory and pro-thrombogenic microparticles. Assessing the impact of mechanical trauma to leukocytes is largely overlooked in comparison to red blood cells and platelets. This review provides an overview of the available literature on the effects of mechanical circulatory support systems on leukocyte phenotype and function. One purpose of this review is to emphasise the importance of studying mechanical trauma to leukocytes to better understand the occurrence of adverse events during mechanical circulatory support.

The Inflammatory Response to Ventricular Assist Devices.

The therapeutic use of ventricular assist devices (VADs) for end-stage heart failure (HF) patients who are ineligible for transplant has increased steadily in the last decade. In parallel, improvements in VAD design have reduced device size, cost, and device-related complications. These complications include infection and thrombosis which share underpinning contribution from the inflammatory response and remain common risks from VAD implantation. An added and underappreciated difficulty in designing a VAD that supports heart function and aids the repair of damaged myocardium is that different types of HF are accompanied by different inflammatory profiles that can affect the response to the implanted device. Circulating inflammatory markers and changes in leukocyte phenotypes receive much attention as biomarkers for mortality and disease progression. However, they are seldom used to monitor progress during and outcomes from VAD therapy or during the design phase for new devices. Even the partial reversal of heart damage associated with heart failure is a desirable outcome from VAD use. Therefore, improved understanding of the interplay between VADs and the recipient's inflammatory response would potentially increase their uptake, improve patient lives, and fuel research related to other blood-contacting medical devices. Here we provide a review of what is currently known about inflammation in heart failure and how this inflammatory profile is altered in heart failure patients receiving VAD therapy.

Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a protein that is secreted immediately upon endothelial injury, and thereby it plays a key role in inflammation via recruitment of leucocytes to the site of inflammation at the beginning and throughout the inflammatory processes. Aim of this study was to develop two separate cell lines displaying either human MCP-1 (HMCP-1) or rabbit MCP-1 (RMCP-1) on their surface. A DNA fragment containing HMCP-1- or RMCP-1-encoding sequence was inserted into a pcDNA plasmid. Escherichia coli cells strain TOP 10F' was separately transformed with the pcDNA/RMCP-1 or /HMCP-1 ligation mixture. Following the cloning and construct verification, human embryonic kidney cell line (HEK 293T) was transfected with either of the linearized plasmids. Plasmid integration into the genomic DNA of HEK 293T cells was verified by polymerase chain reaction (PCR). HMCP-1 and RMCP-1 expression was evaluated at RNA and protein levels by real-time PCR and flow cytometry, respectively. PCR products of the expected sizes were amplified from the chromosomal DNA of transfected HEK 293T cells, i.e. 644 bp for H-MCP1 and 737 bp for RMCP-1 constructs. Real-time PCR revealed that the copy numbers of RMCP1 and HMCP1 mRNA per cell were 294 and 500, respectively. Flow cytometry analysis indicated 85% for RMCP-1 and 87% for HMCP-1 expression levels on the surface of transfected cells, when compared with an isotype control. The experiments thus confirmed that the MCP-1 genes were integrated into the HEK 293T genomic DNA and the encoded proteins were stably expressed on the cell surface.

Disruption of SOX6 gene using CRISPR/Cas9 technology for gamma-globin reactivation: An approach towards gene therapy of ß-thalassemia.

Elevation of Hemoglobin F ameliorates symptoms of ß-thalassemia, a common autosomal recessive disorder. The transcription factor SOX6 plays a key role in the γ to ß-globin gene switching. In the current investigation, a mutation was induced using the CRISPR/Cas9 technology in the binding domain region of SOX6 to reactivate γ-globin expression. Three CRISPR/Cas9 cassettes were provided, whose single-guide RNAs targeted different regions in the SOX6 gene-binding domain. After transfection of K562 cells with CRISPR a, b and c, and subsequent erythroid differentiation, the indel percentage of the cells was about 30%, 25%, and 24%, respectively. Relative quantification showed that the γ-globin mRNA level increased to 1.3-, 2.1-, and 1.1-fold in the cells treated with CRISPR/Cas9 a, b, and c, respectively, compared with untreated cells. Our results show that mutation induction in the binding site of the SOX6 gene leads to γ-globin reactivation. These findings support the idea that CRISPR interrupts the SOX6 binding site, and, as a result, SOX6 is incapable of binding the γ-globin promoter. In conclusion, SOX6 disruption could be considered as a therapeutic approach for ß-thalassemia treatment. CRISPR/Cas9 was selected for this purpose as it is the most rapidly evolving technology.

Ovine Leukocyte Microparticles Generated by the CentriMag Ventricular Assist Device In Vitro.

Ventricular assist devices (VADs) are a life-saving form of mechanical circulatory support in heart failure patients. However, VADs have not yet reached their full potential due to the associated side effects (thrombosis, bleeding, infection) related to the activation and damage of blood cells and proteins caused by mechanical stress and foreign materials. Studies of the effects of VADs on leukocytes are limited, yet leukocyte activation and damage including microparticle generation can influence both thrombosis and infection rates. Therefore, the aim was to develop a multicolor flow cytometry assessment of leukocyte microparticles (LMPs) using ovine blood and the CentriMag VAD as a model for shear stress. Ovine blood was pumped for 6 h in the CentriMag and regular samples analyzed for hemolysis, complete blood counts and LMP by flow cytometry during three different pump operating conditions (low flow, standard, high speed). The high speed condition caused significant increases in plasma-free hemoglobin; decreases in total leukocytes, granulocytes, monocytes, and platelets; increases in CD45+ LMPs as well as two novel LMP populations: CD11bbright /HLA-DR- and CD11bdull /HLA-DR+ , both of which were CD14- /CD21- . CD11bbright /HLA-DR- LMPs appeared to respond to an increase in shear magnitude whereas the CD11bdull /HLA-DR+ LMPs significantly increased in all pumping conditions. We propose that these two populations are released from granulocytes and T cells, respectively, but further research is needed to better characterize these two populations.

The effect of ventricular assist device-associated biomaterials on human blood leukocytes.

Ventricular assist devices (VADs) are an effective bridging or destination therapy for patients with advanced stage heart failure. These devices remain susceptible to adverse events including infection, bleeding, and thrombus; events linked to the foreign body response. Therefore, the biocompatibility of all biomaterials used is crucial to the success of medical devices. Biomaterials common in VADs-DLC: diamond-like carbon coated stainless steel; Sap: single-crystal sapphire; SiN: silicon nitride; Ti: titanium alloy; and ZTA: zirconia-toughened alumina-were tested for their biocompatibility through incubation with whole human blood for 2 h with mild agitation. Blood was then removed and used for: complete cell counts; leukocyte activation and death, and the production of key inflammatory cytokines. All were compared to time 0 and an un-exposed 2 h sample. Monocyte numbers were lower after exposure to DLC, SiN, and ZTA and monocytes showed evidence of activation with DLC, Sap, and SiN. Neutrophils and lymphocytes were unaffected. This approach allows comprehensive analysis of the potential blood damaging effects of biomaterials. Monocyte activation by DLC, Sap, ZTA, and SiN warrants further investigation linking effects on this cell type to unfavorable inflammatory/thrombogenic responses to VADs and other blood handling devices. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1730-1738, 2018.

Shear Stress-Induced Total Blood Trauma in Multiple Species.

The common complications in heart failure patients with implanted ventricular assist devices (VADs) include hemolysis, thrombosis, and bleeding. These are linked to shear stress-induced trauma to erythrocytes, platelets, and von Willebrand factor (vWF). Novel device designs are being developed to reduce the blood trauma, which will need to undergo in vitro and in vivo preclinical testing in large animal models such as cattle, sheep, and pig. To fully understand the impact of device design and enable translation of preclinical results, it is important to identify any potential species-specific differences in the VAD-associated common complications. Therefore, the purpose of this study was to evaluate the effects of shear stress on cells and proteins in bovine, ovine, and porcine blood compared to human. Blood from different species was subjected to various shear rates (0-8000/s) using a rheometer. It was then analyzed for complete blood counts, hemolysis by the Harboe assay, platelet activation by flow cytometry, vWF structure by immunoblotting, and function by collagen binding activity ELISA (vWF : CBA). Overall, increasing shear rate caused increased total blood trauma in all tested species. This analysis revealed species-specific differences in shear-induced hemolysis, platelet activation, and vWF structure and function. Compared to human blood, porcine blood was the most resilient and showed less hemolysis, similar blood counts, but less platelet activation and less vWF damage in response to shear. Compared to human blood, sheared bovine blood showed less hemolysis, similar blood cell counts, greater platelet activation, and similar degradation of vWF structure, but less impact on its activity in response to shear. The shear-induced effect on ovine blood depended on whether the blood was collected via gravity at the abattoir or by venepuncture from live sheep. Overall, ovine abattoir blood was the least resilient in response to shear and bovine blood was the most similar to human blood. These results lay the foundations for developing blood trauma evaluation standards to enable the extrapolation of in vitro and in vivo animal data to predict safety and biocompatibility of blood-handling medical devices in humans. We advise using ovine venepuncture blood instead of ovine abattoir blood due to the greater overall damage in the latter. We propose using bovine blood for total blood damage in vitro device evaluation but multiple species could be used to create a full understanding of the complication risk profile of new devices. Further, this study highlights that choice of antibody clone for evaluating platelet activation in bovine blood can influence the interpretation of results from different studies.

Isolation of Mesenchymal Stromal Cells From Peripheral Blood of ST Elevation Myocardial Infarction Patients.

Bone marrow mesenchymal stromal cells (MSCs) have shown therapeutic potential in the treatment of myocardial infarction patients. However, bone marrow requires invasive harvesting techniques. Therefore, the aim was to carry out a feasibility study of using autologous peripheral blood (PB) as a source for MSCs and platelet lysate (PL), a potential novel therapeutic intervention in acute ST elevation myocardial infarction (STEMI) patients. Autologous PL and MSCs were prepared from STEMI patient and healthy control blood. MSCs were analyzed by trilineage differentiation and flow cytometry. PB MSCs were isolated from 83% of patients (n = 6) but not from controls. The use of PL was feasible in the first passage but not in subsequent ones due to volume. To conclude, PB is a promising alternative to bone marrow. It negates the need for invasive harvesting techniques, and reduces hemorrhagic risk in this patient population routinely managed with anticoagulant and antiplatelet agents.

Quantification methods for human and large animal leukocytes using DNA dyes by flow cytometry.

Ovine and bovine blood is used heavily within the development of blood-handling medical devices, such as heart pumps (left ventricular assist devices, LVADs), for which blood cell damage needs to be monitored during in vitro testing. Hematology analyzers provide cell counts but no information about cell viability. The anthraquinone DNA dyes CyTRAK Orange™ and DRAQ7™ have practical and spectral properties rendering them suitable for multicolor assays. Compared to other DNA dyes such as Vybrant Dyecycle, CyTRAK Orange enables a faster staining protocol and does not require incubation at +37°C. Compared to traditional viability dyes such as propidium iodide and 7AAD, DRAQ7's unique spectral profile of excitation in both blue and red lasers and far-red emission enables identification of dual positive dead cell events and frees up detectors for use with other reagents. CyTRAK Orange and DRAQ7 could be used in combination with absolute counting bead standards to provide cell counts and viability but the combination of these dyes has previously only been used for microscopy on rodent cells. The purpose of this study was to evaluate the use of these dyes in combination in large animal blood samples for flow cytometry. A viability and cell counting protocol for bovine, ovine, and human leukocytes using CyTRAK Orange and DRAQ7 was prepared. Four different counting bead standards were evaluated using the Navios and FACSAria cytometers and compared to counts obtained from hematology analyzers. CyTRAK Orange successfully detected CD45(+) leukocytes in all species. The DRAQ7 single-stained dead cell counts correlated well with the CyTRAK Orange/DRAQ7 double-stained dead cell counts in human and bovine blood, but not in ovine blood, which could be related to the blood source. In conclusion, for human and bovine blood, this method works well for viability counts using different flow cytometers and bead standards. © 2016 International Society for Advancement of Cytometry.

Evaluation of Four Veterinary Hematology Analyzers for Bovine and Ovine Blood Counts for In Vitro Testing of Medical Devices.

Small affordable automated hematology analyzers that produce rapid and accurate complete blood cell counts are a valuable tool to researchers developing blood-handling medical devices, such as ventricular assist devices, for in vitro safety assessments. In such studies, it is common to use the blood of large animals such as cattle and sheep. However, the commercially available instruments have not been evaluated for their ability to measure the blood counts of these animals. In this study, we compare, for the first time, four veterinary analyzers for blood counts on bovine and ovine blood samples. We look at ease of use, repeatability and agreement with a view to inform researchers of the benefits of these instruments in routine measurement of ovine and bovine bloods during in vitro testing. Complete blood cell counts and a three-part differential (granulocytes, monocytes, and lymphocytes) were measured by each of the instruments, and the results compared to those obtained from two additional analyzers used in a reference laboratory. Repeatability and agreement were evaluated using the Bland-Altman method; bias and 95% limits of agreement between the instruments, and between the instruments and two reference instruments, were used to evaluate instrument performance. In summary, there are advantages and disadvantages with all instruments. Of the four instruments tested, the repeatability and agreement was fairly similar for all instruments except one instrument which cannot be recommended for bovine or ovine blood counts.

Altered Th17/Treg Ratio in Recurrent Miscarriage after Treatment with Paternal Lymphocytes and Vitamin D3: a Double-Blind Placebo-Controlled Study.

BACKGROUND: Recurrent miscarriage (RM) affects 2-5% of pregnant women. Paternal lymphocyte immunotherapy is a common treatment for RM patients but the outcome has not been consistent. Therefore, combined therapy with other immunosuppressive drugs such as 1a, 25-dihydroxy-vitamin-D3 (vitamin D3) may improve the outcome. OBJECTIVES: To investigate the effect of vitamin D3 on the balance of two essential T cells subsets, T helper (Th) 17 and T regulatory (Treg) cells, which contribute to the immune tolerance during pregnancy. METHODS: The expression levels of CD4 and forkhead box protein 3 (FOXP3) in Treg cells, and the expression levels of CD4 and IL-17 in Th17 cells, were evaluated pre- and 3 months post-immunotherapy in RM patients treated with a combination of paternal lymphocytes and vitamin D3 compared with RM patients receiving lymphocyte immunotherapy alone. RESULTS: Vitamin D3 therapy decreased the frequency of Th17 cells in addition to reducing the Th17/Treg ratio in peripheral blood of RM patients compared with the control group (p<0.05). CONCLUSION: Considering that RM patients have a higher Th17/Treg ratio in peripheral blood, vitamin D3 may be a candidate therapeutic approach in this disease.

The CentriMag centrifugal blood pump as a benchmark for in vitro testing of hemocompatibility in implantable ventricular assist devices.

Implantable ventricular assist devices (VADs) have proven efficient in advanced heart failure patients as a bridge-to-transplant or destination therapy. However, VAD usage often leads to infection, bleeding, and thrombosis, side effects attributable to the damage to blood cells and plasma proteins. Measuring hemolysis alone does not provide sufficient information to understand total blood damage, and research exploring the impact of currently available pumps on a wider range of blood cell types and plasma proteins such as von Willebrand factor (vWF) is required to further our understanding of safer pump design. The extracorporeal CentriMag (Thoratec Corporation, Pleasanton, CA, USA) has a hemolysis profile within published standards of normalized index of hemolysis levels of less than 0.01 g/100 L at 100 mm Hg but the effect on leukocytes, vWF multimers, and platelets is unknown. Here, the CentriMag was tested using bovine blood (n = 15) under constant hemodynamic conditions in comparison with a static control for total blood cell counts, hemolysis, leukocyte death, vWF multimers, microparticles, platelet activation, and apoptosis. The CentriMag decreased the levels of healthy leukocytes (P < 0.006), induced leukocyte microparticles (P < 10(-5) ), and the level of high molecular weight of vWF multimers was significantly reduced in the CentriMag (P < 10(-5) ) all compared with the static treatment after 6 h in vitro testing. Despite the leukocyte damage, microparticle formation, and cleavage of vWF multimers, these results show that the CentriMag is a hemocompatible pump which could be used as a standard in blood damage assays to inform the design of new implantable blood pumps.

The effect of shear stress on the size, structure, and function of human von Willebrand factor.

Clinical outcomes from ventricular assist devices (VADs) have improved significantly during recent decades, but bleeding episodes remain a common complication of long-term VAD usage. Greater understanding of the effect of the shear stress in the VAD on platelet aggregation, which is influenced by the functional activity of high molecular weight (HMW) von Willebrand factor (vWF), could provide insight into these bleeding complications. However, because VAD shear rates are difficult to assess, there is a need for a model that enables controlled shear rates to first establish the relationship between shear rates and vWF damage. Secondly, if such a dependency exists, then it is relevant to establish a rapid and quantitative assay that can be used routinely for the safety assessment of new VADs in development. Therefore, the purpose of this study was to exert vWF to controlled levels of shear using a rheometer, and flow cytometry was used to investigate the shear-dependent effect on the functional activity of vWF. Human platelet-poor plasma (PPP) was subjected to different shear rate levels ranging from 0 to 8000/s for a period of 6 h using a rheometer. A simple and rapid flow cytometric assay was used to determine platelet aggregation in the presence of ristocetin cofactor as a readout for vWF activity. Platelet aggregates were visualized by confocal microscopy. Multimers of vWF were detected using gel electrophoresis and immunoblotting. The longer PPP was exposed to high shear, the greater the loss of HMW vWF multimers, and the lower the functional activity of vWF for platelet aggregation. Confocal microscopy revealed for the first time that platelet aggregates were smaller and more dispersed in postsheared PPP compared with nonsheared PPP. The loss of HMW vWF in postsheared PPP was demonstrated by immunoblotting. Smaller vWF platelet aggregates formed in response to shear stress might be a cause of bleeding in patients implanted with VADs. The methodological approaches used herein could be useful in the design of safer VADs and other blood handling devices. In particular, we have demonstrated a correlation between the loss of HMW vWF, analyzed by immunoblotting, with platelet aggregation, assessed by flow cytometry. This suggests that flow cytometry could replace conventional immunoblotting as a simple and rapid routine test for HMW vWF loss during in vitro testing of devices.