A biomimetic hydrogel functionalized with adipose ECM components as a microenvironment for the 3D culture of human and murine adipocytes.

The lack of relevant in vitro models for adipose tissue makes necessary the development of a more physiological environment providing spatial and chemical cues for the effective maturation of adipocytes. We developed a biofunctionalized hydrogel with components of adipose extracellular matrix: collagen I, collagen VI, and the cell binding domain of fibronectin and we compared it to usual 2D cultures on plastic plates. This scaffold allowed 3D culture of mature adipocytes from the preadipocytes cell lines 3T3-L1 and 3T3-F442A, as well as primary Human White Preadipocytes (HWP), acquiring in vivo-like organization, with spheroid shaped adipocytes forming multicellular aggregates. The size of these aggregates increased with time up to 120 µm in diameter after 4 weeks of maturation, with good viability. Significantly higher lipogenic activity (up to 20-fold at day 28 for HWP cultures) and differentiation rates were also observed compared to 2D. Gene expression analyses highlighted earlier differentiation and complete maturation of 3D HWP compared to 2D, reinforced by the expression of Perilipin protein after 21 days of nutrition. This increase in adipocytes phenotypic and genotypic markers made this scaffold-driven culture as a robust adipose 3D model. Retinoic acid inhibition of lipogenesis in HWP or isoprenalin and caffeine induction of lipolysis performed on mouse 3T3-F442A cells, showed higher doses of molecules than typically used in 2D, underlying the physiologic relevance of this 3D culture system. Biotechnol. Bioeng. 2017;114: 1813-1824. © 2017 Wiley Periodicals, Inc.

Salvage therapy with gemcitabine, vinorelbine, and pegylated liposomal doxorubicin for relapsed or refractory pediatric Hodgkin lymphoma. Results of a retrospective series of four children.

While the vast majority of pediatric Hodgkin lymphoma (HL) is curable, the prognosis of early relapses and refractory diseases remains poor. Recently, the combination of gemcitabine, vinorelbine, and pegylated liposomal doxorubicin (GVD) displayed encouraging results on adults with relapsed/refractory diseases. Here, we retrospectively report the results of the GVD salvage regimen on four pediatric patients of our ward with a heavily pretreated relapsed/refractory HL. Three of them had nodular sclerosis subtype. They achieved a complete remission after two, three, or four courses of GVD and received high-dose chemotherapy followed by a stem-cell transplantation (auto-auto, auto-allo, and auto). They have been in sustained remission respectively for almost 2, 4, and 5 years. The fourth patient had a lymphocyte-depleted subtype which was initially diagnosed as anaplastic large cell lymphoma. After four courses of GVD, his disease stabilized then progressed again. In total, 15 cycles of GVD were applied to the patients. The toxicity of each course of GVD was mainly hematologic. No cardiotoxicity occurred despite a prior exposure to anthracyclines and radiotherapy. Thus, the GVD combination seems to be an effective pre-transplant rescue treatment for pediatric patients.

Genipin-cross-linked layer-by-layer assemblies: biocompatible microenvironments to direct bone cell fate.

The design of biomimetic coatings capable of improving the osseointegration of bone biomaterials is a current challenge in the field of bone repair. Toward this end, layer-by-layer (LbL) films composed of natural components are suitable candidates. Chondroitin sulfate A (CSA), a natural glycosaminoglycan (GAG), was used as the polyanionic component because it promotes osteoblast maturation in vivo. In their native state, GAG-containing LbL films are generally cytophobic because of their low stiffness. To stiffen our CSA-based LbL films, genipin (GnP) was used as a natural cross-linking agent, which is much less cytotoxic than conventional chemical cross-linkers. GnP-cross-linked films display an original combination of microscale topography and tunable mechanical properties. Structural characterization was partly based on a novel donor/acceptor Förster resonance energy transfer (FRET) couple, namely, FITC/GnP, which is a promising approach for further inspection of any GnP-cross-linked system. GnP-cross-linked films significantly promote adhesion, proliferation, and early and late differentiation of preosteoblasts.

Is pegfilgrastim safe and effective in congenital neutropenia? An analysis of the French Severe Chronic Neutropenia registry.

AIMS: To examine the efficacy and safety of pegfilgrastim in patients with congenital neutropenia (CN). METHODS: Seventeen patients enrolled in the French Severe CN Register received pegfilgrastim. RESULTS: Median age at pegfilgrastim introduction was 19.1 years (range 3.9-52.3 years). In 14 cases pegfilgrastim replaced GCSF (filgrastim or lenograstim), after a median of 6.9 years of GCSF therapy. The dose of pegfilgrastim was usually one full vial per injection (except in five children, who received 1/6 to 1/2 a vial), resulting in a dose of between 50 and 286 microg/kg. The pegfilgrastim schedule ranged from two injections every 7 days to one injection every 30 days, with treatment-free periods. The median interval between the first and last dose of pegfilgrastim was 0.8 years (0.01-4.1 years). The absolute neutrophil count tended to increase more strongly on pegfilgrastim than on GCSF, but the difference was not statistically significant. During pegfilgrastim therapy, a severe infection occurred in two patients and recurrent ENT infections in two other patients. Bone pain was reported by nine patients, anemia and thrombocytopenia occurred in one patient (WHO grade III), chronic urticaria occurred in one patient (WHO grade III), and a single pegfilgrastim injection was followed by respiratory distress and death 15 days later in a patient with GDSIb. At the last update, 10 patients had stopped receiving pegfilgrastim and seven patients were still receiving pegfilgrastim. CONCLUSION: Compared to conventional GCSF, pegfilgrastim is more difficult to use in congenital neutropenia, with more frequent adverse events and sometimes poor efficacy.

Hyaluronan hydrogel: an appropriate three-dimensional model for evaluation of anticancer drug sensitivity.

The extracellular polysaccharide hyaluronan (HA) controls cell migration, differentiation and proliferation, and contributes to the invasiveness of human cancers. In order to investigate the sensitivity of cancer cells to antimitotic agents, we developed a cross-linked HA hydrogel, a three-dimensional matrix in which cells can invade and grow. We have studied three cell lines (SA87, NCI-H460 and H460M), from primary tumors and metastases, that migrated into the HA hydrogel and proliferated giving rise to clusters and colonies. Concurrently, we studied the growth of these cell lines in a usual monolayer culture system. In these two models, increasing concentrations of doxorubicin and 5-fluorouracil were evaluated for their ability to inhibit tumor cell growth and colony formation. Taken together, our data suggest that the cancer cells were more resistant in the three-dimensional model than in monolayer cell systems. The antimitotic drugs were efficient after 24h of treatment in the monolayer cultures, whereas they were significantly efficient only after one week of incubation in the HA hydrogels. Herein, we show that this cross-linked matrix provides a three-dimensional model particularly appropriate for investigating mechanisms involved in cancer cell line sensitivity to antimitotic drugs.

Synergistic influence of topomimetic and chondroitin sulfate-based treatments on osteogenic potential of Ti-6Al-4V.

We combined topographical and chemical surface modifications of Ti-6Al-4V (TA6V) to improve its osteogenic potential. By acid-etching, we first generated topomimetic surface features resembling, in size and roughness, bone cavities left by osteoclasts. Next, we coated these surfaces with biomimetic Layer-by-Layer films (LbL), composed of chondroitin sulfate A and poly-l-lysine that were mechanically tuned after a post-treatment with genipin. The structural impact of each surface processing step was thoroughly inspected. The desired nano/microrough topographies of TA6V were maintained upon LbL deposition. Whereas no significant promotion of adhesion and proliferation of MC3T3-E1 preosteoblasts were detected after independent or combined modifications of the topography and the chemical composition of the substrates, osteogenic maturation was promoted when both surface treatments were combined, as was evidenced by significant long-term matrix mineralization. The results open promising route toward improved osseointegration of titanium-based implants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1988-2000, 2016.

Reticulated hyaluronan hydrogels: a model for examining cancer cell invasion in 3D.

The extracellular polysaccharide hyaluronan (HA) controls cell migration, differentiation and proliferation, and contributes to the invasiveness of human cancers. The roles of HA cell surface receptors and hyaluronidases (HAses) in this process are still controversial. In order to investigate their involvement in cancer pathogenesis, we developed a reticulated HA hydrogel, a three-dimensional matrix in which cells can invade and grow. We have studied thirteen cell lines, from primary tumors or metastases, that migrated into the HA hydrogel and proliferated giving rise to clusters and colonies. The number of colonies, which reflects tumor cell invasiveness, ranged from 7 to 193 after 5 days of culture. Invasion was dependent on the production of HAse as well as other factors. Optimal colonization occurred when cells released HAse, lacked HA-binding sites and did not secrete HA. Moreover, we describe for the first time a HAse activity at physiological pH that may be responding to the confinement of the enzyme in a three-dimensional structure. We show here that this reticulated matrix provides a three-dimensional model for investigating mechanisms involved in malignant invasion.

Survival of cord blood haematopoietic stem cells in a hyaluronan hydrogel for ex vivo biomimicry.

Haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs) grow in a specified niche in close association with the microenvironment, the so-called 'haematopoietic niche'. Scaffolds have been introduced to overcome the liquid culture limitations, mimicking the presence of the extracellular matrix (ECM). In the present study the hyaluronic acid scaffold, already developed in the laboratory, has been used for the first time to maintain long-term cultures of CD34⁺ haematopoietic cells obtained from human cord blood. One parameter investigated was the impact on ex vivo survival of CD34⁺ cord blood cells (CBCs) on the hyaluronic acid surface, immobilized with peptides containing the RGD motif. This peptide was conjugated by coating the hyaluronan hydrogel and cultured in serum-free liquid phase complemented with stem cell factor (SCF), a commonly indispensable cytokine for haematopoiesis. Our work demonstrated that these hyaluronan hydrogels were superior to traditional liquid cultures by maintaining and expanding the HPCs without the need for additional cytokines, and a colonization of 280-fold increment in the hydrogel compared with liquid culture after 28 days of ex vivo expansion.

Liposomal daunorubicin (Daunoxome) and polyethylated glycol conjugated asparaginase (PEG-ASPA) in children with relapsed and refractory acute lymphoblastic leukemia treated on compassionate basis.

BACKGROUND: Daunoxome (DNX) is an encapsulated form of daunorubicin in liposomal vesicles with suggested better pharmacokinetics, pharmacodynamics and lesser cardio toxicity than the free form. Polyethyl glycol asparaginase (PEG-ASPA), a modified form of L-asparaginase, has better activity and lesser immunogenicity than its native molecule. AIM: To evaluate on a compassionate basis, the combination of Daunoxome and PEG-ASPA, as regard to efficacy and toxicity, as a salvage treatment in refractory/relapsed childhood ALL. METHODS: The combination of these 2 drugs were used in 9 multiple relapsed or refractory ALL children on the basis of: DNX weekly 100 mg/m2 D1, 8, 15. PEG-ASPA single, 2500IU/m2 dose D15. Vinca alkaloids and corticosteroids were associated. RESULTS: Median hospital stay was 4 days (0-55). COMPLICATIONS: Neutropenia grade IV n=4, grade III infection n=6, grade II cardiac toxicity n=1, grade III allergy, grade III hemostasis disorders, thrombosis, n=1 respectively. All patients achieved complete remission (CR) except one who died from disease progression. 8 patients were subjected to hematopoietic stem cell transplantatation (HSCT). Two patients of them are alive and well, 4 died from transplant related causes and 2 from disease progression. CONCLUSION: Salvage treatment containing DNX and PEG-ASPA, of small sample childhood ALL with very advanced disease, is feasible as regard toxicity and response rate allowing to HSCT and possible cure.