Bone Marrow Platelet Research Articles

Lung Cancer Detection Using Neural Net

Abstract: Leukemia is a sort of blood threatening development which happens due touncommon development in WBCs (white platelets) in bone marrow of human body. Leukemia can be appointed serious leukemia and progressing leukemia, affected severely and causes malignant growth. In the vast majority of the cases early in which serious leukemia turns out to be astoundingly fast however steady leukemia creates slowly .Among all kinds of kinds of developments, cell breakdown in the lungs is the most overwhelming contamination having the most critical passing rate. Dealt with tomography investigates are utilized for ID of lung hurt as it gives unmistakable image of harmful development in the body and tracks its development. Picture dealing with systems are utilized generally in clinical fields for beginning stage affirmation of cell breakdown in the lungs. The calculation for cell breakdown in the lungs disclosure is proposed utilizing strategies, for example, focus disconnecting for picture pre-dealing with followed by division of lung area of interest utilizing numerical morphological endeavors. Mathematical parts are dealt with from the eliminated district of interest and used to bundle CT channel pictures into typical and abnormal by utilizing support vector machine. Further both the sorts have two sub arrangements lymphocytic and myeloid. In this paper, we will explore different picture dealing with and Man-made intelligence procedures used for request of leukemia area and endeavor to focus in on advantages and limitations of different similar investigates to summarize an result which will be valuable for various experts.

Perspectives of vitamin C upregulating regulatory T cells in the era of thrombopoietin receptor agonists for immune thrombocytopenia

Background Immune thrombocytopenia (ITP) is a rare disorder involving excessive peripheral destruction of platelets and inadequate bone marrow platelet production due to autoimmune reactions against megakaryocytes. Diagnosis is usually by the exclusion of primary causes; its rarity would only permit trial and error therapy experience. Historically, for ITP, oral ascorbic acid (AA) gave inconsistent results. However, unlike other nutrients, AA is found to have an exceptionally tight intestinal absorption restriction mandating intravenous delivery for any therapeutic purposes. Recently, as there is more pre-clinical evidence of AA restoring regulatory T cells (Tregs) by robust demethylation, the historical oral route was likely to be the “bottle neck” restricting effective blood levels. During this era of thrombopoietin receptor agonists (TPO-RAs), planned tapering is often required upon discontinuation but unplanned discontinuations, for example, due to side effects, are more problematic. Moreover, combinations with TPO-RAs are currently being tried using steroids and rituximab except rituximab is inappropriate during pandemics. Notably, with the indirect (but prognostically relevant) action of TPO-RAs on Tregs, boosting Treg functions by adding other Treg active agents, for example, steroids or AA, seems appropriate, especially if without added toxicity upon combining. Conclusions With updated pharmacokinetic concepts, it is timely to repeat AA clinical trials for ITP but using intravenous administration. Initially, this may be on compassionate grounds for its probable role as an adjunct to TPO-RAs for unsuccessful tapering and, especially, for all unplanned discontinuations of TPO-RAs. Such off-label usage of AA is justified because of AA's robust pre-clinical evidence on demethylation and initial clinical experience (despite the inappropriate administrative route). Moreover, moderate intravenous AA dosages have one of the best safety profiles, let alone its eminent affordability. Admittedly, after the initial clinical experience, more systematic trials on AA are required, especially for the most appropriate dosage range and its efficacy as monotherapy.

Immature platelet levels correlate with disease activity and predict treatment response of thrombocytopenia in lupus patients.

The immature platelet fraction (IPF) represents recently produced platelets in bone marrow and this parameter is increased in patient with primary immune thrombocytopenia (ITP). We investigated the associations between IPF and absolute immature platelet count (AIPC), and clinical parameters in systemic lupus erythematosus (SLE), which has more complex pathological mechanisms than in primary ITP. Patients with SLE were retrospectively reviewed at the University of Tokyo Hospital from May, 2012 to January, 2021. The correlations between clinical parameters and the number of immature platelets were assessed with Spearman's rank correlation coefficients. A multiple logistic regression model was used to identify the independent clinical parameters for IPF and AIPC. The difference in the distribution of time for a complete response (CR) after prednisolone (PSL) administration was also evaluated by log-rank test. A total of 282 SLE patients were enrolled, and 12.41% of those patients showed thrombocytopenia. IPF correlated with clinical parameters such as platelet count (r = -0.58), AIPC (r = 0.64) and systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) (r = 0.24). SLEDAI-2K [odds ratio (OR) (per unit increase), 1.07; 95% CI, 1.013 - 1.13] and thrombocytopenia (OR, 32.23; 95% CI, 11.072 - 93.80) were independent clinical parameters to account for IPF increase. IPF correlated with the number of bone marrow megakaryocytes (n = 19, r = 0.57). Notably, the probability of CR in response to PSL in AIPC-high patients was higher than in AIPC-low patients (hazard ratio, 4.62; 95% CI, 1.07 - 20.02). IPF correlated with disease activity of SLE and represented platelet production in the bone marrow, whereas AIPC predicted a rapid response to steroids in thrombocytopenic patients with SLE.

Megakaryocyte Cytoskeletal Proteins in Platelet Biogenesis and Diseases.

Thrombopoiesis governs the formation of blood platelets in bone marrow by converting megakaryocytes into long, branched proplatelets on which individual platelets are assembled. The megakaryocyte cytoskeleton responds to multiple microenvironmental cues, including chemical and mechanical stimuli, sustaining the platelet shedding. During the megakaryocyte's life cycle, cytoskeletal networks organize cell shape and content, connect them physically and biochemically to the bone marrow vascular niche, and enable the release of platelets into the bloodstream. While the basic building blocks of the cytoskeleton have been studied extensively, new sets of cytoskeleton regulators have emerged as critical components of the dynamic protein network that supports platelet production. Understanding how the interaction of individual molecules of the cytoskeleton governs megakaryocyte behavior is essential to improve knowledge of platelet biogenesis and develop new therapeutic strategies for inherited thrombocytopenias caused by alterations in the cytoskeletal genes.

Secretomes differ between peripheral blood and bone marrow-derived platelets

Purpose: Platelet rich plasmas and bone marrow aspirates are commonly used in orthobiologics for their pro-proliferative and immunomodulating characteristics via platelet degranulation and cell secretions. While platelets are derived from megakaryocytes in the bone marrow, no attention has been paid to the potential benefits of bone marrow platelets and whether or not their contents differ from aging platelets in peripheral blood. The aim of our in vitro study was to characterize the difference in cytokines release from platelets derived from the peripheral blood and bone marrow over an extended time-course. Methods: In the present study, prepared leukocyte-poor peripheral blood-derived platelets in plasma (LPP) and leukocyte-poor bone marrow platelets in plasma (BMP) were derived from six donors, stimulated with calcium chloride, incubated at 37°C and sampled at Day 0, Day 3 and Day 6. At each time point, 15 growth and immunomodulatory factors were quantitated in LPP and BMP. Results: The results illustrate that platelets derived from the bone marrow have a unique secretome profile compared to those of peripheral blood. Of the 15 cytokines quantitated, we found significant differences in M-CSF, HGF, IL-4, IL-10, IRAP and Arginase-1 levels. Each of these cytokines have anti-inflammatory roles with important implications in monocyte polarization. Conclusions: Bone marrow-derived platelets may be useful as a stand-alone orthobiologic or as an effective adjunct to autologous cell therapies where anti-inflammatory and anabolic processes are desired especially with respect to monocyte function, such as in the treatment of osteoarthritis.

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

Background aimsPlatelet-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. MethodsIn 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-1β, interferon gamma, IL-4, IL-10, IL-1 receptor antagonist protein, IL-12p40 and arginase-1. ResultsThe 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. ConclusionsUltimately, 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.

RETRACTED: Embedded web system and expression of vascular endothelial growth factor in children with acute leukemia

This work proposes the realization classification of intense leukemia utilizing BP neural organization calculation and platelet picture extraction. Leukemia is a malignant growth of the platelets delivered by the bone marrow, known as the excess of white platelets. The specific reason for leukemia is, at this point, unclear. However, it has been confirmed that leukemia (lymphocytes or bone marrow cells) is grouped by the type of blood cells affected and how rapidly disease progression (acute leukemia, chronic leukemia). The focus of this study is Acute Myeloid Leukemia (AML). These leukemia kinds can be analyzed by examining infinitesimal platelet pictures and including the expansion in the bone marrow platelets. Yet, its cost is too high regarding time, energy, and amount. Likewise, manual tallying can prompt common blunders in diagnostics. The back spread neural organization calculation is utilized to extricate Acute Lymphocytic Leukemia (ALL) properties and AML platelets. Finally, this proposed Vacuole Imaging Technique (VICT) means system-based segmentation and leukemia imaging based on current state detection and image clustering transformation. The proposed vacuole image classification technology (VICT) method is used to act faster and perform better in predicting inaccuracy in the affected area. Digital image processing is used to identify the type of leukemia. Experimental results suggest that the proposed work achieves an average accuracy of 86.66 percent in classifying the type of acute leukemia.

Thrombosis in Patients with Immune Thrombocytopenia ,Review of Literature

Introduction ITP is characterized by low platelet count due to immune mediated destruction and bleeding tendency. However, during last few decades thromboembolic events have been reported in patients with ITP. This review is done to study the reported cases of thromboembolic phenomenon in patient with ITP in an attempt to assess the patient characteristics and to understand the underlying mechanism. Methods We searched google Scholar, PubMed about cases with ITP and thrombosis the summary is presented in the following table (Table 1). Results Around 30 reported cases of ITP with thrombotic events were identified and a total of 36 events were recognized in last 10 years. The ages ranged from 3 years to 81 years with a mean of 51 years. Most of the patients were young and middle aged (18-65 years of age), meanwhile around 9 patients were elderly (age > 65 years). Only 3 cases were observed in pediatric age. Almost equal incidence in both genders was recognized. Half of the patient had chronic ITP while in the rest it was diagnosed less than a year. 20 out of 36 (55.6%) events happened at platelet count less than 100*10^9. While 16 events were reported with platelet count higher than this or unknown. Majority of the patients (around 64%) developed arterial events while fewer developed venous thrombosis. For treatment, most of the patients (44%) were not receiving any particular treatment for ITP at the time of thrombotic event. While 6 events (17%) happened while being treated with IVIG and 10 events (28%') happened while on TPO-RA. Only 3 patients were treated with corticosteroids prior to the event. In patients treated with TPO-RAs arterial and venous events were almost similar (57% vs 43% respectively) while majority of the events happened at lower than normal platelet count (7/10 events). Almost half of the patients had one or more underlying risk factor predisposing to atherosclerosis and thrombosis. Most of the patients were treated appropriately for the events with either antiplatelet agents or anticoagulation while simultaneously treatment for ITP was given. Corticosteroids were most frequently used for ITP during the episode followed by IVIG (52% and 28% of total treated patients respectively). Only 1 patient was treated with TPO-RA after the event for low platelet counts while others received other treatments (Rituximab, Danazol and splenectomy). Discussion Thrombosis is a complex process involving arteries and veins. Accelerated atherosclerosis and plaque rupture is the underlying event for arterial thrombosis. While in venous thrombosis immobility and procoagulant states are the main factors. Immune thrombocytopenia is characterized by immune mediated destruction and impaired production of platelets predisposing to bleeding mostly. However, it is a unique pathological process that is linked to both bleeding and thrombosis. Multiple factors predispose patients to thrombosis in ITP. The patients with chronic and active disease are particularly at risk of paradoxical thrombosis due to accelerated atherosclerosis as in other autoimmune conditions, predisposing to arterial thrombotic events. Active disease is also characterized by increased turnover of platelets in bone marrow and higher levels of circulating platelets microparticles (PMPs) which promote thrombin formation and promote venous thrombosis. The patients treated with IVIG and TPO-RA are at higher risk as compared to other forms of treatment. IVIG is used in acute states as it prevents the destruction of platelets but simultaneously promotes thrombosis by increasing blood viscosity and thrombin production. TPO-RAs are agents which mimic the action of thrombopoietin on megakaryocytes promoting their growth and differentiation and increasing platelet production. Increasing platelet count above the normal target might contribute to thrombosis however megakaryocyte activation itself leads to increased risk of thrombosis, despite low platelet count. In patients with ITP and thrombotic events, judicious use of antiplatelet therapy and anticoagulation is indicated along with simultaneous therapy directed at improving platelet count. Conclusion Patient with active ITP are predisposed to thrombosis in addition to bleeding. A treating physician needs to be vigilant to diagnose early the events and then to institute proper use of antiplatelets and anticoagulation along with therapy directed at ITP. Figure Disclosures No relevant conflicts of interest to declare.

Cellular and Molecular Mechanisms of Environmental Pollutants on Hematopoiesis.

Hematopoiesis is a complex and intricate process that aims to replenish blood components in a constant fashion. It is orchestrated mostly by hematopoietic progenitor cells (hematopoietic stem cells (HSCs)) that are capable of self-renewal and differentiation. These cells can originate other cell subtypes that are responsible for maintaining vital functions, mediate innate and adaptive immune responses, provide tissues with oxygen, and control coagulation. Hematopoiesis in adults takes place in the bone marrow, which is endowed with an extensive vasculature conferring an intense flow of cells. A myriad of cell subtypes can be found in the bone marrow at different levels of activation, being also under constant action of an extensive amount of diverse chemical mediators and enzymatic systems. Bone marrow platelets, mature erythrocytes and leukocytes are delivered into the bloodstream readily available to meet body demands. Leukocytes circulate and reach different tissues, returning or not returning to the bloodstream. Senescent leukocytes, specially granulocytes, return to the bone marrow to be phagocytized by macrophages, restarting granulopoiesis. The constant high production and delivery of cells into the bloodstream, alongside the fact that blood cells can also circulate between tissues, makes the hematopoietic system a prime target for toxic agents to act upon, making the understanding of the bone marrow microenvironment vital for both toxicological sciences and risk assessment. Environmental and occupational pollutants, therapeutic molecules, drugs of abuse, and even nutritional status can directly affect progenitor cells at their differentiation and maturation stages, altering behavior and function of blood compounds and resulting in impaired immune responses, anemias, leukemias, and blood coagulation disturbances. This review aims to describe the most recently investigated molecular and cellular toxicity mechanisms of current major environmental pollutants on hematopoiesis in the bone marrow.

A case series review of patients with Thrombocytopenia and Absent-Radii syndrome (TARS) and their management during pregnancy

Bleeding diatheses due to platelet-related disorders can present challenges to treating clinicians, especially in the context of peri- and post-partum patients in the obstetric setting. Thrombocytopenia and Absent-Radii syndrome (TARS) is an inherited disorder characterized by reduced bone marrow platelet production, skeletal deformities affecting radii and other limbs; cardiac, renal, and other heterogeneous anomalies may occur. It is caused by the co-inheritance of a microdeletion and a nucleotide polymorphism in the RBM8A gene on chromosome 1. Bleeding phenotype is more severe than platelet numbers which might predict especially in infants but improves with age. There is minimal literature regarding the impact of pregnancy and puerperium. We describe the management of three pregnancies in the hematology-obstetrics clinic. As platelet counts normally decrease through pregnancy, close monitoring is required in TARS. No major bleeding was seen antenatally but two required platelet transfusions during labor. No other treatment definitely improves bleeding, although case reports of steroids claim variable success. Tranexamic acid may be helpful, and thrombopoietin agonists represent a potential future option.

Pathogenesis of immune thrombocytopenia in common variable immunodeficiency.

Immune Thrombocitopenic Purpura (ITP) is an autoimmune disease characterized by antibody-mediated platelet destruction and variable reduced platelet production. Besides antibody-mediated platelet destruction, new pathogenic mechanisms have been reported to be involved in reducing platelet count. Among these, desialylation is one of the most recent and innovative mechanisms that has been found to be implied, at least in part, in non-antibody mediated platelet clearance. Common Variable Immunodeficiency (CVID) is the most common Primary Immunodeficiency seen in clinical practice. About 25-30% of CVID patients are affected by autoimmune manifestation, among which ITP is the most common. Little is know about pathophysiological mechanisms that lead to ITP in CVID. Given the poor antibody production typical of CVID patients, we aimed at verifying whether platelet desialylation could be responsible for CVID associated thrombocytopenia. According to our results, we may suggest that in CVID patients, ITP is due to a decreased bone marrow platelets production, rather than an increased peripheral platelet destruction, which is more common in patients with primary ITP. An increased platelet desialylation does not appear to be implicated in the thrombocytopenia secondary to CVID, while it is implicated in the pathogenesis of primary ITP. Nevertheless an intriguing aspect has emerged from this study: regardless the presence of thrombocytopenia, the majority of CVID patients present a double platelet population as far as desialylation concerns, whilst no one of the healthy donors and of the patients with primary ITP shows a similar characteristic.

Trp53 regulates platelets in bone marrow via the PI3K pathway.

The p53 gene is well known as a key tumor suppressor gene; it is vital for hematopoietic stem cell differentiation and growth. In the present study, the change of platelets (PLTs) in p53 knockout mice (p53-/- mice) was investigated. The peripheral blood cell subsets and PLT parameters in p53-/-mice were compared with those in age-matched p53+/+ mice. Bleeding time as well as the alteration of PLT levels, were analyzed with the PLT marker CD41 antibody using flow cytometry. The results revealed that the number of PLTs in p53-/- mice was significantly lower than that in p53+/+ mice. Bleeding time was prolonged in the peripheral blood of p53-/- mice compared with that of p53+/+ mice. Furthermore, the related gene expression of the PI3K signaling pathway in the bone marrow of p53-/- mice was shown to be associated with plateletogenesis. PI3K inhibitor (LY294002) was also used to treat p53-/- mice, and the results demonstrated that LY294002 revert the change of PLTs in these mice. In summary, PLTs were altered in p53-/- mice, and the PI3K signaling pathway was involved in that process, suggesting that the p53-dependent PI3K signaling pathway is involved in thrombocytopenia or PLT diseases. PLT number is reduced in p53 deficiency; however, this reduction could be reverted by inhibiting the PI3K pathway.

Transplanting of Adipose Derived Stem Cells Up-Regulate CD4+CD25+Foxp3+ T Cells in a Murine Model of Passive Chronic Immune Thrombocytopenia

Background: Immune thrombocytopenia (ITP) is an autoimmune disorder in which antibodies and/or T cells lead to enhanced peripheral platelet destruction and reduced bone marrow platelet production. The dysregulated CD4+CD25+Foxp3+ T regulatory cells (Tregs) play a critical role in the pathophysiology of ITP. Human adipose-derived mesenchymal stem cells (hADSCs) were recently found to suppress effector T cell responses and to have beneficial effects in various immune disorders. However, it is not clear how ADSCs could affect ITP. This study is aimed to investigate the therapeutic effect of ADSCs on the treatment of ITP mice and explore the underlying mechanisms. Methods: A passive immune thrombocytopenia animal model was induced by injection of the monoclonal antiplatelet antibody MWReg30, and ADSC injection was performed on the 2nd day after ITP induction. PLT counts have been measured by an automatic blood cell counter daily after ADSC transplant for 6 days. The frequency of Tregs in the blood was analyzed by flow cytometry. mRNAs and proteins of Foxp3, TGF-β1 and IL-10 in the spleen, thymus and lymph nodes were detected by RT-PCR and western blotting respectively. Findings: Transplant of ADSCs on ITP mice led to increased PLT counts, an up-regulation of Tregs in the blood, and an up-regulation of Foxp3, TGF-β1 and IL-10 mRNA and protein in the spleen, thymus and lymph nodes as compared with the untreated ITP mice. Conclusion: ADSCs transplant may be an available method in therapy of ITP. ADSCs transplant may affect proliferation and function of Tregs in ITP, and Foxp3, TGF-β1 and IL-10 may be involved in this process. Funding: This work was supported by the Youth Program of National Natural Science Founds (8150091), The Doctoral Start Project of Natural Science Foundation of Guangdong Province(2015A030310022) and the Youth Program of Natural Science Founds of Hunan Province (2018JJ3472). Declaration of Interest: The authors declare that they have no conflict of interest. Ethical Approval: All experimental procedures conformed to the Guidelines of Animal Experiments from the Chinese Committee of Medical Ethics and were approved by the Commission of Animal Research, Sun Yat-sen University Medical Center. Raw human aspirates from patients undergoing lipectomy were collected after obtaining informed consent according to procedures approved by the Ethics Committee of the Chinese Academy of Medical Sciences and the First Affiliated Hospital of South China University.

Impact of Itga2-Gp6-double collagen receptor deficient mice for bone marrow megakaryocytes and platelets.

The two main collagen receptors on platelets, GPVI and integrin α2β1, play an important role for the recognition of exposed collagen at sites of vessel injury, which leads to platelet activation and subsequently stable thrombus formation. Both receptors are already expressed on megakaryocytes, the platelet forming cells within the bone marrow. Megakaryocytes are in permanent contact with collagen filaments in the marrow cavity and at the basal lamina of sinusoids without obvious preactivation. The role of both collagen receptors for megakaryocyte maturation and thrombopoiesis is still poorly understood. To investigate the function of both collagen receptors, we generated mice that are double deficient for Gp6 and Itga2. Flow cytometric analyses revealed that the deficiency of both receptors had no impact on platelet number and led to the expected lack in GPVI responsiveness. Integrin activation and degranulation ability was comparable to wildtype mice. By immunofluorescence microscopy, we could demonstrate that both wildtype and double-deficient megakaryocytes were overall normally distributed within the bone marrow. We found megakaryocyte count and size to be normal, the localization within the bone marrow, the degree of maturation, as well as their association to sinusoids were also unaltered. However, the contact of megakaryocytes to collagen type I filaments was decreased at sinusoids compared to wildtype mice, while the interaction to type IV collagen was unaffected. Our results imply that GPVI and α2β1 have no influence on the localization of megakaryocytes within the bone marrow, their association to the sinusoids or their maturation. The decreased contact of megakaryocytes to collagen type I might at least partially explain the unaltered platelet phenotype in these mice, since proplatelet formation is mediated by these receptors and their interaction to collagen. It is rather likely that other compensatory signaling pathways and receptors play a role that needs to be elucidated.

Percutaneous piercing cortical bone at fracture ends and local injection of autologous concentrated bone marrow aspirate and platelet rich plasma for treatment of delayed fracture union

Objective To evaluate the clinical efficacy of percutaneous piercing cortical bone at fracture ends and local injection of autologous concentrated bone marrow aspirate (cBMA) and platelet rich plasma (PRP) in the treatment of delayed fracture union. Methods A retrospective study was performed on the clinical data of 34 patients with delayed fracture union who had been treated at Department of Orthopaedics, Luoyang Zhenggu Hospital of Henan Province from April 2014 to February 2017. They were 26 males 8 females, aged from 18 to 63 years (average, 36.7 years). There were 15 femoral, 11 tibial, 5 humerus and 3 ulnar fractures. According to the Weber-Crech classification, 14 cases belonged to the type of rich blood supply-active end hypertrophy and 20 to the type of poor blood supply and malnutrition. There were 28 closed and 6 open fractures. Of this cohort, 21 cases had their primary fixation, 7 had revision once, 4 had revision twice, and 2 had revision more than thrice; 19 cases had intramedullary fixation, 12 had plate fixation and 3 had external fixation. Autologous iliac bone grafting was performed previously in 19 cases. Kirschner wire was used under fluoroscopy to pierce percutaneously the cortical bone at fracture ends; bone marrow blood and peripheral venous blood were collected to prepare autologous cBMA and PRP which were to be injected locally at the fracture ends after mixture. At the last follow-up, the patients were evaluated by visual analogue scale (VAS), SF-36 health survey summary scores and modified Radiographic Union Score for Tibia (RUST). Results The patients were followed up for 12 to 42 months (average, 21.5 months). Thirty-one patients healed after surgery, yielding a healing rate of 91.2%. The healing time averaged 5.3 months (from 4 to 10 months). No patient reported such complications as infection or neurovascular injury. At the last follow-up, the VAS scores of the 34 patients were 1.3±0.6 points, significantly lower than the preoperative 5.4±3.6 points (P<0.05); the postoperative SF-36 scores 74.8±16.5 points, significantly increased than the preoperative 44.8±15.6 scores (P<0.05); the RUST scores 3.3±0.4 points, significantly higher than the preoperative 1.6±0.8 points (P<0.05). Conclusions Minimally invasive percutaneous piercing of K-wire on the cortical bone at the fracture ends may result in fluid leakage and restart bone healing process. At the same time, local injection of autologous cBMA and PRP may increase the number and concentration of local bone marrow mesenchymal stem cells and growth factors, promoting fracture healing. Key words: Fractures, bone; Bone marrow; Platelet-rich plasma; Surgical procedures, minimally invasive; Delayed healing

Abstract 29: Chronic Stress Predisposes to Thrombosis by Abnormal Megakaryopiesis: Protective Effect of Apocynin

Introduction: Psychological stress (e.g. anxiety and depression) has been identified as an important trigger of acute coronary syndromes (ACS), as a consequence of enhanced coagulation and of hyper-reactive platelets. Changes in redox balance, alteration of genes regulating antioxidant systems, including NADPH oxidase, and increased production of reactive oxygen species (ROS) have been measured in both chronic stress and ACS. However, the mechanisms by which chronic stress affects platelet activation and predisposes to thrombosis are not well known. Hypothesis: We hypothesized that Apocynin, an inhibitor of NADPH oxidase influences the alteration of megakaryopoiesis and activation of platelets induced by chronic stress in mice. Methods and Results: We show the NADPH/NADP + ratio in bone marrow (BM) of mice exposed to forced swimming for 4 days (5 min twice/day) is markedly reduced compared to control mice, and that Apocynin treatment (2.4 mg/ml in drinking water for 4 days) prevents this alteration. Chronic stress leads to an abnormal megakaryopoiesis increasing the number of BM megakaryocytes (MKs) and affecting circulating platelets. MKs of stressed mice show an advanced maturation state (e.g. nuclear/cytoplasmic ratios and expression of CD42d), and an enhanced ability to produce ROS. Interestingly, a higher number of large and reticulated platelets with marked functional activation (e.g. integrin α IIb β3 and P-selectin expression, and platelet/leukocyte aggregates) is detected after chronic stress. In addition, Apocynin prevents ROS MKs generation and decreases the total number of MKs without affecting the percentage of CD42d + cells. Finally, the inhibitor of NADPH oxidase activity reduces the hyper-activation of platelets and the enhanced susceptibility to FeCl3-induced arterial thrombosis in stressed mice. Conclusion: Apocynin treatment, reducing ROS generation in MKs, restores the physiological bone marrow megakaryopoiesis and platelet behaviour, and it prevents the detrimental effect of chronic stress on atherothrombosis. These data suggest a potential use of NADPH oxidase inhibitors in the occurrence of thrombosis associated with chronic stress. Studies in human will verify the clinical impact of these findings.

Mechanism of Mitochondria-mediated Pathway in the Platelet Apoptosis Resulted from Immune Bone Marrow Failure

To explore the mechamisms of mitochondria-mediated pathway in apoptosis of platelets resulted from in immune induced bone marrow failure. Thirty C57BL/6 mice were randomly divided into 3 groups (10 mice in each group): normal group, model group, cyclosporine A(CsA) group. Mouse model of immune bone marrow failure were established. After mouse model was successfully established, the mice in normal group and model group were given saline orally, the mice in CsA group was treated with CsA orally. Blood routine examination of mice in each group was performed by automatic blood cell analyzer; the mitochondrial membrane potential(ΔΨm), cytochrome C(Cyt C), phosphatidylserine (PS), Ca2+ were measured by flow cytometry; expression of BAX, BAK, caspase-3, caspase-8, caspase-9 was detected by using Western blot method, the changes of bone marrow platelet ultrastructure were observed under transmission electron microscope. Compared with normal group, the platelet count of model group decreased significantly, while the level of ΔΨm, caspase-3, caspase-8, caspase-9 significantly decreased, the level of Cyt C, PS, Ca2+, BAX, BAK increased significantly (P<0.05). Compared with the model group, the platelet count of CsA group increased obviously, while the level of ΔΨm, caspase-3, caspase-8, caspase-9 of CsA group increased significantly, the level of Cyt C, PS, Ca2+, BAX, BAK of CsA group decreased significantly (P<0.05). Electron microscopy showed that compared with the model group, platelet damage in CsA group were alleviated. mitochondrial pathway plays an important role in the reduction of platelet resulted from immune bone marrow failure.

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs.

Translation of cell therapy into clinical practice: validation of an application procedure for bone marrow progenitor cells and platelet rich plasma.

Tissue regeneration can be improved by local application of autologous bone marrow derived progenitor cells (BMSC) and platelet rich plasma (PRP). However, there is a lack of standardized application procedures for clinical use. Therefore, a technique in accordance with the guidelines for advanced therapies medical products of the European Medicine Agency was developed and established. In detail, a process for the isolation and formulation of autologous bone marrow cells (BMC) and PRP in a clinical setting was validated. To investigate the influence of storage time and temperature on gel formation and gel stability, different concentrations of BMC were stored with and without additional platelets, thrombin and fibrinogen and analyzed over a period of 28 days. In addition, cell vitality using a live-dead staining and migration ability of human mesenchymal stem cells (hMSC) in the gel clot was investigated. For an optimized stable gel clot, human BMC and PRP should be combined with 10% to 20% fibrinogen (9 mg/mL to 18 mg/mL) and 5% to 20% thrombin (25 I.E. to 100 I.E.). Both freshly prepared and stored cells for 1 to 7 days had a stable consistence over 28 days at 37°C. Different platelet concentrations did not influence gel clot formation. The ratio of living cells did not decrease significantly over the observation period of 5 days in the live-dead staining. The study identified an optimal gel texture for local application of BMC and PRP. Seeded hMSC could migrate therein and were able to survive to initiate a healing cascade.

Transplantation of culture-expanded bone marrow cells and platelet rich plasma in lower limb lengthening for short stature patients

Transplantation of culture-expanded bone marrow cells and platelet rich plasma in lower limb lengthening for short stature patients