Biphasic Calcium Phosphate and Activated Carbon Microparticles in a Plasma Clot for Bone Reconstruction and In Situ Drug Delivery: A Feasibility Study.

The development of bone-filling biomaterials capable of delivering in situ bone growth promoters or therapeutic agents is a key area of research. We previously developed a biomaterial constituting biphasic calcium phosphate (BCP) microparticles embedded in an autologous blood or plasma clot, which induced bone-like tissue formation in ectopic sites and mature bone formation in orthotopic sites, in small and large animals. More recently, we showed that activated carbon (AC) fiber cloth is a biocompatible material that can be used, due to its multiscale porosity, as therapeutic drug delivery system. The present work aimed first to assess the feasibility of preparing calibrated AC microparticles, and second to investigate the properties of a BCP/AC microparticle combination embedded in a plasma clot. We show here, for the first time, after subcutaneous (SC) implantation in mice, that the addition of AC microparticles to a BCP/plasma clot does not impair bone-like tissue formation and has a beneficial effect on the vascularization of the newly formed tissue. Our results also confirm, in this SC model, the ability of AC in particle form to adsorb and deliver large molecules at an implantation site. Altogether, these results demonstrate the feasibility of using this BCP/AC/plasma clot composite for bone reconstruction and drug delivery.

Long-Term Fate and Efficacy of a Biomimetic (Sr)-Apatite-Coated Carbon Patch Used for Bone Reconstruction.

Critical bone defect repair remains a major medical challenge. Developing biocompatible materials with bone-healing ability is a key field of research, and calcium-deficient apatites (CDA) are appealing bioactive candidates. We previously described a method to cover activated carbon cloths (ACC) with CDA or strontium-doped CDA coatings to generate bone patches. Our previous study in rats revealed that apposition of ACC or ACC/CDA patches on cortical bone defects accelerated bone repair in the short term. This study aimed to analyze in the medium term the reconstruction of cortical bone in the presence of ACC/CDA or ACC/10Sr-CDA patches corresponding to 6 at.% of strontium substitution. It also aimed to examine the behavior of these cloths in the medium and long term, in situ and at distance. Our results at day 26 confirm the particular efficacy of strontium-doped patches on bone reconstruction, leading to new thick bone with high bone quality as quantified by Raman microspectroscopy. At 6 months the biocompatibility and complete osteointegration of these carbon cloths and the absence of micrometric carbon debris, either out of the implantation site or within peripheral organs, was confirmed. These results demonstrate that these composite carbon patches are promising biomaterials to accelerate bone reconstruction.

Repurposing the Bis-Biguanide Alexidine in Combination with Tyrosine Kinase Inhibitors to Eliminate Leukemic Stem/Progenitor Cells in Chronic Myeloid Leukemia.

BACKGROUND & AIMS: In CML, Leukemic Stem Cells (LSCs) that are insensitive to Tyrosine Kinase Inhibitors are responsible for leukemia maintenance and relapses upon TKI treatment arrest. We previously showed that downregulation of the BMI1 polycomb protein that is crucial for stem/progenitor cells self-renewal induced a CCNG2/dependent proliferation arrest leading to elimination of Chronic Myeloid Leukemia (CML) cells. Unfortunately, as of today, pharmacological inhibition of BMI1 has not made its way to the clinic. METHODS: We used the Connectivity Map bioinformatic database to identify pharmacological molecules that could mimick BMI1 silencing, to induce CML cell death. We selected the bis-biguanide Alexidin (ALX) that produced a transcriptomic profile positively correlating with the one obtained after BMI silencing in K562 CML cells. We then evaluated the efficiency of ALX in combination with TKI on CML cells. RESULTS: Here we report that cell growth and clonogenic activity of K562 and LAMA-84 CML cell lines were strongly inhibited by ALX. ALX didn't modify BCR::ABL1 phosphorylation and didn't affect BMI1 expression but was able to increase CCNG2 expression leading to autophagic processes that preceed cell death. Besides, ALX could enhance the apoptotic response induced by any Tyrosine Kinase Inhibitors (TKI) of the three generations. We also noted a strong synergism between ALX and TKIs to increase expression of caspase-9 and caspase-3 and induce PARP cleavage, Bad expression and significantly decreased Bcl-xL family member expression. We also observed that the blockage of the mitochondrial respiratory chain by ALX can be associated with inhibition of glycolysis by 2-DG to achieve an enhanced inhibition of K562 proliferation and clonogenicity. ALX specifically affected the differentiation of BCR::ABL1-transduced healthy CD34+ cells but not of mock-infected healthy CD34+ control cells. Importantly, ALX strongly synergized with TKIs to inhibit clonogenicity of primary CML CD34+ cells from diagnosed patients. Long Term Culture of Initiating Cell (LTC-IC) and dilution of the fluorescent marker CFSE allowed us to observe that ALX and Imatinib (IM) partially reduced the number of LSCs by themselves but that the ALX/IM combination drastically reduced this cell compartment. Using an in vivo model of NSG mice intravenously injected with K562-Luciferase transduced CML cells, we showed that ALX combined with IM improved mice survival. CONCLUSIONS: Collectively, our results validate the use of ALX bis-biguanide to potentiate the action of conventional TKI treatment as a potential new therapeutic solution to eradicate CML LSCs.

Effects of Fatty Acid Metabolites on Adipocytes Britening: Role of Thromboxane A2.

Obesity is a complex disease highly related to diet and lifestyle and is associated with low amount of thermogenic adipocytes. Therapeutics that regulate brown adipocyte recruitment and activity represent interesting strategies to fight overweight and associated comorbidities. Recent studies suggest a role for several fatty acids and their metabolites, called lipokines, in the control of thermogenesis. The purpose of this work was to analyze the role of several lipokines in the control of brown/brite adipocyte formation. We used a validated human adipocyte model, human multipotent adipose-derived stem cell model (hMADS). In the absence of rosiglitazone, hMADS cells differentiate into white adipocytes, but convert into brite adipocytes upon rosiglitazone or prostacyclin 2 (PGI2) treatment. Gene expression was quantified using RT-qPCR and protein levels were assessed by Western blotting. We show here that lipokines such as 12,13-diHOME, 12-HEPE, 15dPGJ2 and 15dPGJ3 were not able to induce browning of white hMADS adipocytes. However, both fatty acid esters of hydroxy fatty acids (FAHFAs), 9-PAHPA and 9-PAHSA potentiated brown key marker UCP1 mRNA levels. Interestingly, CTA2, the stable analog of thromboxane A2 (TXA2), but not its inactive metabolite TXB2, inhibited the rosiglitazone and PGI2-induced browning of hMADS adipocytes. These results pinpoint TXA2 as a lipokine inhibiting brown adipocyte formation that is antagonized by PGI2. Our data open new horizons in the development of potential therapies based on the control of thromboxane A2/prostacyclin balance to combat obesity and associated metabolic disorders.

In vivo effectiveness of carbonated calcium-deficient hydroxyapatite-coated activated carbon fiber cloth on bone regeneration.

We have previously shown that activated carbon fiber cloth (ACC) either uncoated or coated with carbonated calcium-deficient hydroxyapatite (CDA), namely ACC and ACC/CDA, were biocompatible in vitro with human osteoblasts. Here we hypothesized that ACC and ACC/CDA could be used as tissue patches in vivo to accelerate wounded bone healing. In a model of rat femoral defect, we have compared spontaneous cortical bone regeneration with regeneration in the presence of ACC and ACC/CDA patches. At Day 7, 14, and 21, bone formation was evaluated using microcomputed tomography, magnetic resonance imaging, and histological analysis. Our results demonstrate first that these ACC tissues are highly biocompatible in vivo, and second that ACC/CDA patches apposition results in the acceleration of bone reconstruction due to a guiding action of the ACC fibers and an osteogenic effect of the CDA phase. We guess that this approach may represent a valuable strategy to accelerate bone regeneration in human.

Activated Carbon Fiber Cloth/Biomimetic Apatite: A Dual Drug Delivery System.

A biomaterial that is both bioactive and capable of controlled drug release is highly attractive for bone regeneration. In previous works, we demonstrated the possibility of combining activated carbon fiber cloth (ACC) and biomimetic apatite (such as calcium-deficient hydroxyapatite (CDA)) to develop an efficient material for bone regeneration. The aim to use the adsorption properties of an activated carbon/biomimetic apatite composite to synthetize a biomaterial to be used as a controlled drug release system after implantation. The adsorption and desorption of tetracycline and aspirin were first investigated in the ACC and CDA components and then on ACC/CDA composite. The results showed that drug adsorption and release are dependent on the adsorbent material and the drug polarity/hydrophilicity, leading to two distinct modes of drug adsorption and release. Consequently, a double adsorption approach was successfully performed, leading to a multifunctional and innovative ACC-aspirin/CDA-tetracycline implantable biomaterial. In a second step, in vitro tests emphasized a better affinity of the drug (tetracycline or aspirin)-loaded ACC/CDA materials towards human primary osteoblast viability and proliferation. Then, in vivo experiments on a large cortical bone defect in rats was carried out to test biocompatibility and bone regeneration ability. Data clearly highlighted a significant acceleration of bone reconstruction in the presence of the ACC/CDA patch. The ability of the aspirin-loaded ACC/CDA material to release the drug in situ for improving bone healing was also underlined, as a proof of concept. This work highlights the possibility of bone patches with controlled (multi)drug release features being used for bone tissue repair.

Combination of PKCδ Inhibition with Conventional TKI Treatment to Target CML Models.

Numerous combinations of signaling pathway blockades in association with tyrosine kinase inhibitor (TKI) treatment have been proposed for eradicating leukemic stem cells (LSCs) in chronic myeloid leukemia (CML), but none are currently clinically available. Because targeting protein kinase Cδ (PKCδ) was demonstrated to eliminate cancer stem cells (CSCs) in solid tumors, we evaluated the efficacy of PKCδ inhibition in combination with TKIs for CML cells. We observed that inhibition of PKCδ by a pharmacological inhibitor, by gene silencing, or by using K562 CML cells expressing dominant-negative (DN) or constitutively active (CA) PKCδ isoforms clearly points to PKCδ as a regulator of the expression of the stemness regulator BMI1. As a consequence, inhibition of PKCδ impaired clonogenicity and cell proliferation for leukemic cells. PKCδ targeting in K562 and LAMA-84 CML cell lines clearly enhanced the apoptotic response triggered by any TKI. A strong synergism was observed for apoptosis induction through an increase in caspase-9 and caspase-3 activation and significantly decreased expression of the Bcl-xL Bcl-2 family member. Inhibition of PKCδ did not modify BCR-ABL phosphorylation but acted downstream of the oncogene by downregulating BMI1 expression, decreasing clonogenicity. PKCδ inhibition interfered with the clonogenicity of primary CML CD34+ and BCR-ABL-transduced healthy CD34+ cells as efficiently as any TKI while it did not affect differentiation of healthy CD34+ cells. LTC-IC experiments pinpointed that PKCδ inhibition strongly decreased the progenitors/LSCs frequency. All together, these results demonstrate that targeting of PKCδ in combination with a conventional TKI could be a new therapeutic opportunity to affect for CML cells.

Proteomic analysis identified LBP and CD14 as key proteins in blood/biphasic calcium phosphate microparticle interactions.

Immediately upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to the biomaterial surface and the protein layer affects both blood cell functions and biomaterial bioactivity. Previously, we reported that 80-200 µm biphasic calcium phosphate (BCP) microparticles embedded in a blood clot, induce ectopic woven bone formation in mice, when 200-500 µm BCP particles induce mainly fibrous tissue. Here, in a LC-MS/MS proteomic study we compared the differentially expressed blood proteins (plasma and blood cell proteins) and the deregulated signaling pathways of these osteogenic and fibrogenic blood composites. We showed that blood/BCP-induced osteogenesis is associated with a higher expression of fibrinogen (FGN) and an upregulation of the Myd88- and NF-κB-dependent TLR4 signaling cascade. We also highlighted the key role of the LBP/CD14 proteins in the TLR4 activation of blood cells by BCP particles. As FGN is an endogenous ligand of TLR4, able to modulate blood composite stiffness, we propose that different FGN concentrations modify the blood clot mechanical properties, which in turn modulate BCP/blood composite osteoactivity through TLR4 signaling. The present findings provide an insight at the protein level, into the mechanisms leading to an efficient bone reconstruction by blood/BCP composites. STATEMENT OF SIGNIFICANCE: Upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to bone substitute surface and this protein layer affects both biomaterial bioactivity and bone healing. Therefore, for the best outcome for patients, it is crucial to understand the molecular interactions between blood and bone scaffolds. Biphasic calcium phosphate (BCP) ceramics are considered as the gold standard in bone reconstruction surgery. Here, using proteomic analyses we showed that the osteogenic properties of 80-200 µm BCP particles embedded in a blood clot is associated with a higher expression of fibrinogen. Fibrinogen upregulates the Myd88- and NF-κB-dependent TLR4 pathway in blood cells and, BCP-induced TLR4 activation is mediated by the LBP and CD14 proteins.

The Rosmarinus Bioactive Compound Carnosic Acid Is a Novel PPAR Antagonist That Inhibits the Browning of White Adipocytes.

Thermogenic brown and brite adipocytes convert chemical energy from nutrients into heat. Therapeutics that regulate brown adipocyte recruitment and activity represent interesting strategies to control fat mass such as in obesity or cachexia. The peroxisome proliferator-activated receptor (PPAR) family plays key roles in the maintenance of adipose tissue and in the regulation of thermogenic activity. Activation of these receptors induce browning of white adipocyte. The purpose of this work was to characterize the role of carnosic acid (CA), a compound used in traditional medicine, in the control of brown/brite adipocyte formation and function. We used human multipotent adipose-derived stem (hMADS) cells differentiated into white or brite adipocytes. The expression of key marker genes was determined using RT-qPCR and western blotting. We show here that CA inhibits the browning of white adipocytes and favors decreased gene expression of thermogenic markers. CA treatment does not affect ß-adrenergic response. Importantly, the effects of CA are fully reversible. We used transactivation assays to show that CA has a PPARα/γ antagonistic action. Our data pinpoint CA as a drug able to control PPAR activity through an antagonistic effect. These observations shed some light on the development of natural PPAR antagonists and their potential effects on thermogenic response.

The adiponectin receptor agonist AdipoRon normalizes glucose metabolism and prevents obesity but not growth retardation induced by glucocorticoids in young mice.

OBJECTIVE: Glucocorticoids (GCs) are highly effective anti-inflammatory and immunosuppressive drugs. However, prolonged GC therapy may cause numerous adverse effects leading to diabetes and obesity, as well as bone disorders such as osteoporosis in adults and growth retardation in children and adolescents. Prevention and care of the GC-induced adverse effects remain challenging. We have previously demonstrated the efficacy of a treatment with a non-peptidic agonist of adiponectin receptors, AdipoRon, to reverse behaviour disorders and fat mass gain induced by long-term GC treatment. In this work, we have established a relevant model of GC-induced growth and metabolic disorders and determined that AdipoRon is a potential therapeutic tool to reverse these metabolic disturbances. METHODS: 5-Week-old mice were treated continuously with or without corticosterone (35 mg/L) in drinking water for seven consecutive weeks. Taking advantage of this mouse model displaying various growth and metabolic disorders, we assayed whether AdipoRon (daily intraperitoneal injection of 1 mg/kg/day for the last 20 days) might prevent the GC-induced adverse effects. The control group was treated with vehicle only. Nutritional behaviors and metabolic parameters were followed-up throughout the treatment. Serum insulin and leptin levels were measured by ELISA. Computed tomography and histological analysis of adipose tissue were assessed at the end of the experimental procedure. RESULTS: We found that GC treatment in young mice resulted in continuously increased body weight gain associated with a food intake increase. Compared to vehicle-, GC-treated mice displayed early major hyperleptinemia (up to 6-fold more) and hyperinsulinemia (up to 20-fold more) maintained throughout the treatment. At the end of the experimental procedure, GC-treated mice displayed bone growth retardation (e.g. femur length 15.1 versus 14.0 mm, P < 0.01), higher abdominal adipose tissue volume (4.1 versus 2.3, P < 0.01) and altered glucose metabolism compared to control mice. Interestingly, AdipoRon prevented GC-induced effects on energy metabolism such as abdominal adiposity, insulinemia and leptinemia. However, AdipoRon failed to counteract bone growth retardation. CONCLUSION: We characterized the very early pathological steps induced by long-term GC in young mice in a relevant model, including growth retardation, fat mass gain and glucose homeostasis dysregulation. The adiponectin system stimulation enabled normalization of the adipose tissue and metabolic features of GC-treated mice. Adiponectin receptor agonists such as AdipoRon might constitute a novel way to counteract some GC-induced adverse effects.

Adjuvant intensity modulated whole-abdominal radiation therapy for high-risk patients with ovarian cancer FIGO stage III: final results of a prospective phase 2 study.

BACKGROUND: To assess late toxicity, quality of life and oncological outcome after consolidative whole abdominal radiotherapy (WART) following cytoreductive surgery and carboplatin/paclitaxel chemotherapy in high risk patients with advanced ovarian cancer FIGO stage III using IMRT (Intensity modulated radiation therapy). METHODS: The OVAR-IMRT-02 study is a multi-center single-arm phase-II-trial. Twenty patients with optimally debulked ovarian cancer stage FIGO III with complete remission after chemotherapy were treated with intensity modulated WART. A total dose of 30 Gy in 20 fractions was applied to the entire peritoneal cavity. Primary endpoint was treatment tolerability; secondary objectives were acute and chronic toxicities, quality of life, rates of therapy disruption/abortion, progression-free survival (PFS) and overall survival (OS). RESULTS: All patients completed treatment and 10/20 patients (50%) reached the final study follow-up of 36 months. Late side effects consisted of °1-°2 lower limb edema (44.5%), with one patient (5.6%) showing °3 edema. Three patients (16.7%) showed elevated gamma-Glutamyltransferase. There were no severe late side effects regarding renal or hepatic function or any gastrointestinal toxicity greater than °2. During WART, mean global health status decreased by 18.1 points (95%-CI: 7.1-29.0), but completely normalized after 6 months. The same trend was observed for the function scale scores. Kaplan-Meier-estimated 1-, 2- and 3-year PFS was 74, 51 and 40%, respectively. 1-, 2- and 3-year OS was 89, 83 and 83%, respectively. CONCLUSIONS: Intensity modulated WART after aggressive surgery and carboplatin/paclitaxel chemotherapy is associated with an acceptable risk of acute and late toxicity and minor impact on long-term quality of life. Together with the promising results for PFS and OS, intensity modulated WART could offer a new therapeutic option for consolidation treatment of patients with advanced ovarian cancer. TRIAL REGISTRATION: The study is registered with ClinicalTrials.gov ( NCT01180504 ). Registered 12 August 2010 - retrospectively registered.

Combination of blood and biphasic calcium phosphate microparticles for the reconstruction of large bone defects in dog: A pilot study.

We previously reported that biphasic calcium phosphate (BCP) microparticles embedded in a blood clot induces ectopic bone formation in mice and repairs a critical femoral defect in rat. The present pilot study aimed to evaluate in dog and in two models of large defects the efficacy of this composite named "blood for reconstruction of bone" (BRB). We show here that BRB is a cohesive biomaterial easy to prepare from dog autologous blood and to mold to fill large bone defects. First in a model of cylindrical femoral condyle defect, the BRB was compared with BCP particles alone. After 8 weeks, this revealed that the amount of mature bone was slightly and significantly higher with BRB than with BCP particles. Second, in a model consisting in a 2 cm-long critical interruptive defect of the ulna, the BRB was compared with autologous bone. After 6 months, we observed that implantation of BRB can induce the complete reconstruction of the defect and that newly formed bone exhibits high regenerative potential. Comparison with the results obtained with autologous bone grafting strongly suggests that the BRB might be an efficient biomaterial to repair large bone defects, as an alternative or in addition to autologous bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1842-1850, 2018.

Macrophage-derived oncostatin M contributes to human and mouse neurogenic heterotopic ossifications.

Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury-induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.

Adjuvant Intensity Modulated Whole-Abdominal Radiation Therapy for High-Risk Patients With Ovarian Cancer (International Federation of Gynecology and Obstetrics Stage III): First Results of a Prospective Phase 2 Study.

PURPOSE: To assess treatment tolerance and toxicity rates of consolidative whole-abdominal radiation therapy (WART) following cytoreductive surgery and carboplatin/paclitaxel chemotherapy in high-risk patients with advanced ovarian cancer (International Federation of Gynecology and Obstetrics stage III) using intensity modulated radiation therapy. METHODS AND MATERIALS: The OVAR-IMRT-02 study is a multicenter, single-arm, phase 2 trial. Twenty patients with optimally debulked ovarian cancer (International Federation of Gynecology and Obstetrics stage III) with complete remission after chemotherapy were treated with intensity modulated WART as a consolidation therapy. A total dose of 30 Gy in 20 fractions of 1.5 Gy was applied to the entire peritoneal cavity. The primary endpoint was treatment tolerability, defined as lack of any Common Terminology Criteria for Adverse Events grade 4 toxicity within 10 weeks after start of treatment; secondary objectives were acute and chronic toxicity, quality of life, rates of therapy disruption and abortion, and progression-free and overall survival. RESULTS: Intensity modulated WART resulted in excellent coverage of the whole peritoneal cavity, with effective sparing of all organs at risk. The primary analysis included all 20 enrolled patients, of whom 19 did not experience Common Terminology Criteria for Adverse Events grade 4 toxicity. Only 1 patient experienced acute grade 4 hematologic toxicity. Thus, the tolerability rate of intensity modulated WART was significantly higher than 70%. No gastrointestinal acute toxicities higher than grade 2 have been observed. During WART, mean global health status decreased by 18.1 points (95% confidence interval 7.1, 29.0). Six weeks after WART, global health status had already increased, with a mean score difference of 4.6 (95% confidence interval -11.1, 20.4) compared with baseline. Similar characteristics were observed for all function scale scores. CONCLUSION: Intensity modulated WART after aggressive surgery and carboplatin/paclitaxel chemotherapy is associated with an acceptable risk of acute toxicity and a treatment tolerability rate significantly higher than 70%. Together with our knowledge about clinical feasibility, meaning excellent coverage of the planning target volume and effective sparing of organs at risk, intensity modulated WART could offer a new therapeutic option for consolidation treatment of patients with advanced ovarian cancer.

Calcium supplementation decreases BCP-induced inflammatory processes in blood cells through the NLRP3 inflammasome down-regulation.

Interaction of host blood with biomaterials is the first event occurring after implantation in a bone defect. This study aimed at investigating the cellular and molecular consequences arising at the interface between whole blood and biphasic calcium phosphate (BCP) particles. We observed that, due to calcium capture, BCP inhibited blood coagulation, and that this inhibition was reversed by calcium supplementation. Therefore, we studied the impact of calcium supplementation on BCP effects on blood cells. Comparative analysis of BCP and calcium supplemented-BCP (BCP/Ca) effects on blood cells showed that BCP as well as BCP/Ca induced monocyte proliferation, as well as a weak but significant hemolysis. Our data showed for the first time that calcium supplementation of BCP microparticles had anti-inflammatory properties compared to BCP alone that induced an inflammatory response in blood cells. Our results strongly suggest that the anti-inflammatory property of calcium supplemented-BCP results from its down-modulating effect on P2X7R gene expression and its capacity to inhibit ATP/P2X7R interactions, decreasing the NLRP3 inflammasome activation. Considering that monocytes have a vast regenerative potential, and since the excessive inflammation often observed after bone substitutes implantation limits their performance, our results might have great implications in terms of understanding the mechanisms leading to an efficient bone reconstruction. STATEMENT OF SIGNIFICANCE: Although scaffolds and biomaterials unavoidably come into direct contact with blood during bone defect filling, whole blood-biomaterials interactions have been poorly explored. By studying in 3D the interactions between biphasic calcium phosphate (BCP) in microparticulate form and blood, we showed for the first time that calcium supplementation of BCP microparticles (BCP/Ca) has anti-inflammatory properties compared to BCP-induced inflammation in whole blood cells and provided information related to the molecular mechanisms involved. The present study also showed that BCP, as well as BCP/Ca particles stimulate monocyte proliferation. As monocytes represent a powerful target for regenerative therapies and as an excessive inflammation limits the performance of biomaterials in bone tissue engineering, our results might have great implications to improve bone reconstruction.

BCL-B (BCL2L10) is overexpressed in patients suffering from multiple myeloma (MM) and drives an MM-like disease in transgenic mice.

Multiple myeloma (MM) evolves from a premalignant condition known as monoclonal gammopathy of undetermined significance (MGUS). However, the factors underlying the malignant transformation of plasmocytes in MM are not fully characterized. We report here that Eµ-directed expression of the antiapoptotic Bcl-B protein in mice drives an MM phenotype that reproduces accurately the human disease. Indeed, with age, Eµ-bcl-b transgenic mice develop the characteristic features of human MM, including bone malignant plasma cell infiltration, a monoclonal immunoglobulin peak, immunoglobulin deposit in renal tubules, and highly characteristic bone lytic lesions. In addition, the tumors are serially transplantable in irradiated wild-type mice, underlying the tumoral origin of the disease. Eµ-bcl-b plasmocytes show increased expression of a panel of genes known to be dysregulated in human MM pathogenesis. Treatment of Eµ-bcl-b mice with drugs currently used to treat patients such as melphalan and VELCADE efficiently kills malignant plasmocytes in vivo. Finally, we find that Bcl-B is overexpressed in plasmocytes from MM patients but neither in MGUS patients nor in healthy individuals, suggesting that Bcl-B may drive MM. These findings suggest that Bcl-B could be an important factor in MM disease and pinpoint Eµ-bcl-b mice as a pertinent model to validate new therapies in MM.

Green laser light irradiation enhances differentiation and matrix mineralization of osteogenic cells.

BACKGROUND AND OBJECTIVE: Low level laser therapy (LLLT) in both infrared and visible light is a therapeutic tool ever more proposed in clinical practice in different fields. The effect of near infrared LLLT has been described in a growing number of scientific publications related to bone tissue healing, both in vitro and in vivo. More recently, green visible light using potassium-titanyl-phosphate KTiOPO4 (KTP, 532 nm) laser has been proposed in dermatology, urology, oral and maxillofacial surgery but has never been tested on bone tissue. The aim of the present work was to perform a preliminary in vitro study to analyze the effects of KTP laser, on the osteogenic differentiation of bone marrow stromal cells (BMSCs). MATERIALS AND METHODS: Using a power meter the first step of this study aimed to evaluate the real power emitted by the KTP laser device and the amount of energy absorbed by culture medium and plastic in order to calculate the appropriate irradiation parameters for cultured cells. Primary bone marrow stromal cells prepared from C57BL/6 mice were cultured and induced to differentiate in the osteogenic lineage in the presence or in the absence of KTP LLLT at a fluence of 4 J/cm(2) three times a week. Specific staining of the cells and the extracellular matrix, microscopic analysis as well as quantitative RT-PCR were used to assess cell proliferation and differentiation. RESULTS: We show here that KTP LLLT enhances the osteogenic differentiation of bone marrow stromal cells and the mineralization of their extracellular matrix. CONCLUSION: Our results highlight that this LLLT experimental protocol with green light (KTP, 532 nm) at 4 J/cm(2) has a positive effect on the osteogenic differentiation of murine bone marrow stromal cells. These preliminary results could be used as a basis to further investigate the effect of this KTP laser protocol on bone tissue engineering models in vivo and in vitro.

Osteogenic Potential of Mesenchymal Stromal Cells Contributes to Primary Myelofibrosis.

Primary myelofibrosis is a myeloproliferative neoplasm that is a precursor to myeloid leukemia. Dysmegakaryopoiesis and extramedullary hematopoiesis characterize primary myelofibrosis, which is also associated with bone marrow stromal alterations marked by fibrosis, neoangiogenesis, and osteomyelosclerosis. In particular, contributions to primary myelofibrosis from mesenchymal stromal cells (MSC) have been suggested by mouse studies, but evidence in humans remains lacking. In this study, we show that bone marrow MSCs from primary myelofibrosis patients exhibit unique molecular and functional abnormalities distinct from other myeloproliferative neoplasms and these abnormalities are maintained stably ex vivo in the absence of leukemic cells. Primary myelofibrosis-MSC overexpressed heparin-binding cytokines, including proinflammatory TGFß1 and osteogenic BMP-2, as well as glycosaminoglycans such as heparan sulfate and chondroitin sulfate. Transcriptome and functional analyses revealed alterations in MSC differentiation characterized by an increased osteogenic potential and a TGFß1 signaling signature. Accordingly, phospho-Smad2 levels were intrinsically increased in primary myelofibrosis-MSC along with enhanced expression of the master bone regulator RUNX2, while inhibition of the endogenous TGFß1 receptor TGFßR1 impaired osteogenic differentiation in these MSCs. Taken together, our results define the source of a critical osteogenic function in primary myelofibrosis that supports its pathophysiology, suggesting that combined targeting of both the hematopoietic and stromal cell compartments in primary myelofibrosis patients may heighten therapeutic efficacy.

Intensity-modulated whole abdomen irradiation following adjuvant carboplatin/taxane chemotherapy for FIGO stage III ovarian cancer : four-year outcomes.

INTRODUCTION: A prospective study to assess toxicity and survival outcomes after intensity-modulated whole-abdominal irradiation (IM-WAI) following surgery and adjuvant intravenous carboplatin/taxane chemotherapy in advanced FIGO stage III ovarian cancer. PATIENTS AND METHODS: Between 2006 and 2009, 16 patients with optimally resected FIGO stage III ovarian cancer, who had received six cycles of adjuvant carboplatin/taxane chemotherapy were treated with consolidation IM-WAI. Radiotherapy was delivered to a total dose of 30 Gy in 1.5-Gy fractions, using step-and-shoot (n = 3) or helical tomotherapy (n = 13). The first 10 patients were treated within a phase I trial; the following patients received the same treatment modality. The target volume included the entire peritoneal cavity, the diaphragm, the liver capsule, and the pelvic and para-aortic node regions. Organs at risk were kidneys, liver, heart, and bone marrow. RESULTS: Median follow-up was 44 months (range 19.2-67.2 months). No grade 4 toxicities occurred during IM-WAI. Common Toxicity Criteria for Adverse Events (CTCAE) grade 3 toxicities were: diarrhea (25 %), leucopenia (19 %), nausea/vomiting (6 %), and thrombocytopenia (6 %). No toxicity-related treatment break was necessary. Small bowel obstruction occurred in a total of 6 patients: in 3 cases (19 %) due to postsurgical adhesions and in 3 cases due to local tumor recurrence (19 %). Median recurrence-free survival (RFS) was 27.6 months (95 % confidence interval, CI = 24-44 months) and median overall survival (OS) was 42.1 months (95 %CI = 17-68 months). The peritoneal cavity was the most frequent site of initial failure. CONCLUSION: Consolidation IM-WAI following surgery and adjuvant chemotherapy is feasible and can be performed with manageable acute and late toxicity. The favorable RFS outcome is promising and justifies further clinical trials.

Effect of G-CSF on the osteoinductive property of a BCP/blood clot composite.

We previously reported that blood clot combined with biphasic calcium phosphate microparticles constitute a biomaterial (BRB) that can repair a bone critical defect in rat and induces subcutaneous bone formation in mice. The granulocyte colony-stimulating factor (G-CSF) is the agent most commonly used in human to enrich blood with hematopoietic stem and progenitor cells (HSPCs) as well as granulocytes (GCs). Moreover, recent data also suggest that it can mobilize mesenchymal stem cells (MSCs). Here, we asked whether the osteoinductive properties of the BRB could be further enhanced by G-CSF, either by replacing normal blood by G-CSF-mobilized blood (BRBe) or by treating the recipient animals with G-CSF. The experiments performed in C57BL/6 mice showed that G-CSF induces a marked increase of circulating HPCs and GCs, but not of MSCs. BRBe prepared with G-CSF-enriched blood induced a slight but significant decrease of subcutaneous bone formation compared to BRB prepared with normal blood. Additional injection of G-CSF to the recipient mice had no significant effect on the bone formation induced by BRB or BRBe. Altogether these results indicate that, in this model of ectopic implantation, cell mobilization induced by G-CSF has a negative effect on the osteoinductive property of this blood/BCP composite.