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.

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.

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.

Treatment of squamous cell carcinoma of the mobile tongue or tongue margins: an interdisciplinary challenge.

BACKGROUND: Standard treatment is surgery with stage dependent postoperative radio(chemo)therapy, however, for organ preservation preoperative radio(chemo)therapy is used as an individual approach. The present analysis was performed to access outcome and toxicity of radiotherapeutical treatment of squamous cell carcinoma of the tongue. PATIENTS AND METHODS: Sixty-six patients (median age 55 years) with cancer of the mobile tongue (n=30) or tongue margins (n=36) treated between 1982 and 2006 were retrospectively analyzed. Treatment consisted of definitive- (n=13, median dose 66 Gy), adjuvant- (n=31, median dose 60 Gy) or neoadjuvant radiotherapy (n=22, median dose 40 Gy) and chemotherapy (n=34) or immunotherapy (n=1). RESULTS: After a median follow-up of 29 months the three- and five-year overall survival (OS) rates were 59% and 46%, respectively. The median OS was 54 months. Forty-two patients achieved complete remission whereas 14 patients showed partial remission. The one- and two-year loco-regional progression-free survival (LRPFS) rates were 76% and 58%, respectively. The median LRPFS time was 36 months. In χ(2)-test, T-stage showed a trend towards impact on local recurrence (Pearson, p=0.082). In multivariate analysis, alcohol consumption (p=0.003) and gender (p=0.031) were prognostic. Grade III/IV acute toxicity was seen in 52% of patients. None of the locally controlled patients reported grade IV or higher late toxicity. CONCLUSION: No statistically significant differences between treatment modalities were found, but one should keep in mind that organ preservation plays a major role for quality of life. None of the locally controlled patients reported grade IV or higher late toxicity. However, tumor recurrence is common, especially in advanced tumor stage. Interdisciplinary concepts, further increasing the chance of tumor control are warranted.

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.

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.

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.

Monocytes differentiation upon treatment with a peptide corresponding to the C-terminus of activated T cell-expressed Tirc7 protein.

Atp6v0a3 gene encodes for two alternative products, Tirc7 and a3 proteins, which are differentially expressed in activated T cells and resorbing osteoclasts, respectively. Tirc7 plays a central role in T cell activation, while a3 protein is critical for osteoclast-mediated bone matrix resorption. Based on the large body of evidences documenting the relationships between T cells and osteoclasts, we hypothesized that the extracellular C-terminus of Tirc7 protein could directly interact with osteoclast precursor cells. To address this issue, we performed the molecular cloning of a mouse Atp6v0a3 cDNA segment encoding the last 40 amino acids of Tirc7 protein, and we used this peptide as a ligand added to mouse osteoclast precursor cells. We evidenced that Tirc7-Cter peptide induced the differentiation of RAW264.7 cells into osteoclast-like cells, stimulated an autocrine/paracrine regulatory loop potentially involved in osteoclastic differentiation control, and strongly up-regulated F4/80 protein expression within multinucleated osteoclast-like cells. Using a mouse bone marrow-derived CD11b(+) cell line, or total bone marrow primary cells, we observed that similarly to Rankl, Tirc7-Cter peptide induced the formation of TRACP-positive large multinucleated cells. At last, using mouse primary monocytes purified from total bone marrow, we determined that Tirc7-Cter peptide induced the appearance of small multinucleated cells (3-4 nuclei), devoid of resorbing activity, and which displayed modulations of dendritic cell marker genes expression. In conclusion, we report for the first time on biological effects mediated by a peptide corresponding to the C-terminus of Tirc7 protein, which interfere with monocytic differentiation pathways.

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.

Phase II study evaluating consolidation whole abdominal intensity-modulated radiotherapy (IMRT) in patients with advanced ovarian cancer stage FIGO III--the OVAR-IMRT-02 Study.

BACKGROUND: The prognosis for patients with advanced FIGO stage III epithelial ovarian cancer remains poor despite the aggressive standard treatment, consisting of maximal cytoreductive surgery and platinum-based chemotherapy. The median time to recurrence is less than 2 years, with a 5-years survival rate of -20-25%. Recurrences of the disease occur mostly intraperitoneally.Ovarian cancer is a radiosensitive tumor, so that the use of whole abdominal radiotherapy (WAR) as a consolidation therapy would appear to be a logical strategy. WAR used to be the standard treatment after surgery before the chemotherapy era; however, it has been almost totally excluded from the treatment of ovarian cancer during the past decade because of its high toxicity. Modern intensity-modulated radiation therapy (IMRT) has the potential of sparing organs at risk like kidneys, liver, and bone marrow while still adequately covering the peritoneal cavity with a homogenous dose.Our previous phase I study showed for the first time the clinical feasibility of intensity-modulated WAR and pointed out promising results concerning treatment tolerance. The current phase-II study succeeds to the phase-I study to further evaluate the toxicity of this new treatment. METHODS/DESIGN: The OVAR-IMRT-02 study is a single-center one arm phase-II trial. Thirty seven patients with optimally debulked ovarian cancer stage FIGO III having a complete remission after chemotherapy will be treated with intensity-modulated WAR as a consolidation therapy.A total dose of 30 Gy in 20 fractions of 1.5 Gy will be applied to the entire peritoneal cavity including the liver surface and the pelvic and para-aortic node regions. Organ at risk are kidneys, liver (except the 1 cm-outer border), heart, vertebral bodies and pelvic bones.Primary endpoint is tolerability; secondary objectives are toxicity, quality of life, progression-free and overall survival. DISCUSSION: Intensity-modulated WAR provides a new promising option in the consolidation treatment of ovarian carcinoma in patients with a complete pathologic remission after adjuvant chemotherapy. Further consequent studies will be needed to enable firm conclusions regarding the value of consolidation radiotherapy within the multimodal treatment of advanced ovarian cancer. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01180504.

Randomized controlled phase I/II study to investigate immune stimulatory effects by low dose radiotherapy in primarily operable pancreatic cancer.

BACKGROUND: The efficiencies of T cell based immunotherapies are affected by insufficient migration and activation of tumor specific effector T cells in the tumor. Accumulating evidence exists on the ability of ionizing radiation to modify the tumor microenvironment and generate inflammation. The aim of this phase I/II clinical trial is to evaluate whether low dose single fraction radiotherapy can improve T cell associated antitumor immune response in patients with pancreatic cancer. METHODS/DESIGN: This trial has been designed as an investigator initiated; prospective randomised, 4-armed, controlled Phase I/II trial. Patients who are candidates for resection of pancreatic cancer will be randomized into 4 arms. A total of 40 patients will be enrolled. The patients receive 0 Gy, 0.5 Gy, 2 Gy or 5 Gy radiation precisely targeted to their pancreatic carcinoma. Radiation will be delivered by external beam radiotherapy using a 6 MV Linac with IMRT technique 48 h prior to the surgical resection. The primary objective is the determination of an active local external beam radiation dose, leading to tumor infiltrating T cells as a surrogate parameter for antitumor activity. Secondary objectives include local tumor control and recurrence patterns, survival, radiogenic treatment toxicity and postoperative morbidity and mortality, as well as quality of life. Further, frequencies of tumor reactive T cells in blood and bone marrow as well as whole blood cell transcriptomics and plasma-proteomics will be correlated with clinical outcome. An interim analysis will be performed after the enrollment of 20 patients for safety reasons. The evaluation of the primary endpoint will start four weeks after the last patient's enrollment. DISCUSSION: This trial will answer the question whether a low dose radiotherapy localized to the pancreatic tumor only can increase the number of tumor infiltrating T cells and thus potentially enhance the antitumor immune response. The study will also investigate the prognostic and predictive value of radiation-induced T cell activity along with transcriptomic and proteomic data with respect to clinical outcome.

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.

Activation of hedgehog signaling inhibits osteoblast differentiation of human mesenchymal stem cells.

Mesenchymal stem cells within the bone are responsible for the generation of osteoblasts, chondrocytes, and adipocytes. In rodents, Indian hedgehog has been shown to play a role in osteoblast differentiation. However, evidence for a direct function of hedgehog (Hh) in human osteoblastic differentiation is missing. Using different models of human mesenchymal stem cells we show that Hh signaling decreases during osteoblast differentiation. This is associated with a decrease in Smoothened expression, a key partner that triggers Hh signaling, and in the number of cells displaying a primary cilium, an organelle necessary for Hh signaling. Remarkably, treatment of human mesenchymal stem cells with sonic hedgehog or two molecules able to activate Hh signaling inhibits osteoblast differentiation. This inhibition is visualized through a decrease in mineralization and in the expression of osteoblastic genes. In particular, activation of Hh signaling induces a decrease in Runx2 expression, a key transcriptional factor controlling the early stage of osteoblast differentiation. Consistently, the activation of Hh signaling during the first days of differentiation is sufficient to inhibit osteoblast differentiation, whereas differentiated osteoblasts are not affected by Hh signaling. In summary, we show here, using various inducers of Hh signaling and mesenchymal stem cells of two different origins, that Hh signaling inhibits human osteoblast differentiation, in sharp contrast to what has been described in rodent cells. This species difference should be taken into account for screening for pro-osteogenic molecules.

Osteoclastic differentiation of mouse and human monocytes in a plasma clot/biphasic calcium phosphate microparticles composite.

We recently demonstrated that blood clotted around biphasic calcium phosphate (BCP) microparticles constituted a composite biomaterial that could be used for bone defect filling. In addition, we showed that mononuclear cells, i.e. monocytes and lymphocytes, play a central role in the osteogenic effect of this biomaterial. Hypothesizing that osteoclast progenitors could participate to the pro-osteogenic effect of mononuclear cells we observed previously, we focus on this population through the study of mouse monocyte/macrophage cells (RAW264.7 cell line), as well as human pre-osteoclastic cells derived from mononuclear hematopoietic progenitor cells (monocytes-enriched fraction from peripheral blood). Using monocyte-derived osteoclast progenitors cultured within plasma clot/BCP microparticles composite, we aimed in the present report at the elucidation of transcriptional profiles of genes related to osteoclastogenesis and to bone remodelling. For both human and mouse monocytes, real-time PCR experiments demonstrated that plasma clot/BCP scaffold potentiated the expression of marker genes of the osteoclast differentiation such as Nfactc1, Jdp2, Fra2, Tracp and Ctsk. By contrast, Mmp9 was induced in mouse but not in human cells, and Ctr expression was down regulated for both species. In addition, for both mouse and human precursors, osteoclastic differentiation was associated with a strong stimulation of VegfC and Sdf1 genes expression. At last, using field-emission scanning electron microscopy analysis, we observed the interactions between human monocytes and BCP microparticles. As a whole, we demonstrated that plasma clot/BCP microparticles composite provided monocytes with a suitable microenvironment allowing their osteoclastic differentiation, together with the production of pro-angiogenic and chemoattractant factors.

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.

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.

Effects of low-level laser therapy on proliferation and differentiation of murine bone marrow cells into osteoblasts and osteoclasts.

BACKGROUND AND OBJECTIVE: Low-Level Laser Therapy (LLLT) has been suggested to improve bone tissue healing. The cellular and molecular mechanisms involved in this effect are still unclear but bone cell proliferation and differentiation alteration have been proposed. The aim of the present study was to investigate, in vitro, the effect of LLLT on bone cell proliferation, osteoblastic and osteoclastic differentiation, both involved in bone remodeling and regeneration. STUDY DESIGN/MATERIALS AND METHODS: Murine bone marrow cells, which contain both osteoblast and osteoclast progenitors, were cultured and induced to differentiate in the absence or in the presence of LLLT. Laser exposition parameters were determined using a powermeter and consisted in an 808 nm infrared wavelength laser light in continuous mode, with an energy density of 4 J/cm(2) administered three times a week. Cell proliferation and differentiation were assessed after specific staining and microscopic analysis of the cultures after various times, as well as by quantitative RT-PCR analysis of a panel of osteoblast and osteoclast markers after nucleic acid extraction. RESULTS: The use of a powermeter revealed that the power emitted by the optical fiber of the laser device was markedly reduced compared to the displayed power. This allowed to adjust the LLLT parameters to a final energy density exposure of 4 J/cm(2). In these conditions, proliferation of bone marrow mesenchymal stem cells as well as osteoclast or osteoblast differentiation of the corresponding progenitors were found similar in control and LLLT conditions. CONCLUSION: Using the present experimental protocol, we concluded that an 808 nm wavelength infrared LLLT does not alter murine bone progenitor cell proliferation and differentiation. Moreover our results confirm the necessary use of a powermeter to fix LLLT protocol parameters.

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.