Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures.

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape.

Conformable hyaluronic acid hydrogel delivers adipose-derived stem cells and promotes regeneration of burn injury.

Injury to the skin from severe burns can cause debilitating physical and psychosocial distress to the patients. Upon healing, deep dermal burns often result in devastating hypertrophic scar formation. For many decades, stem cell-based therapies have shown significant potential in improving wound healing. However, current cell delivery methods are often insufficient to maintain cell viability in a harmful burn wound environment to promote skin regeneration. In this study, we developed an enhanced approach to deliver adipose-derived stem cells (ASCs) for the treatment of burn wounds, using an in-situ-formed hydrogel system comprised of a hyperbranched poly(ethylene glycol) diacrylate (HB-PEGDA) polymer, a commercially available thiol-functionalized hyaluronic acid (HA-SH) and a short RGD peptide. Stable hydrogels with tunable swelling and mechanical properties form within five minutes under physiological conditions via the Michael-type addition reaction. Combining with RGD peptide, as a cell adhesion motif, significantly alters the cellular morphology, enhances cell proliferation, and increases the paracrine activity of angiogenesis and tissue remodeling growth factors and cytokines. Bioluminescence imaging of luciferase+ ASCs indicated that the hydrogel protected the implanted cells from the harmful wound environment in burns. Hydrogel-ASC treatment significantly enhanced neovascularization, accelerated wound closure and reduced the scar formation. Our findings suggest that PEG-HA-RGD-based hydrogel provides an effective niche capable of augmenting the regenerative potential of ASCs and promoting burn wound healing. STATEMENT OF SIGNIFICANCE: Burn injury is one of the most devastating injures, and patients suffer from many complications and post-burn scar formation despite modern therapies. Here, we designed a conformable hydrogel-based stem cell delivery platform that allows rapid in-situ gelation upon contact with wounds. Adipose-derived stem cells were encapsulated into a PEG-HA-RGD hydrogels. Introducing of RGD motif significantly improved the cellular morphology, proliferation, and secretion of angiogenesis and remodeling cytokines. A deep second-degree burn murine model was utilized to evaluate in-vivo cell retention and therapeutic effect of the hydrogel-ASC-based therapy on burn wound healing. Our hydrogel remarkably improved ASCs viability in burn wounds and the hydrogel-ASC treatment enhanced the neovascularization, promoted wound closure, and reduced scar formation.

Identification of compound CA-5f as a novel late-stage autophagy inhibitor with potent anti-tumor effect against non-small cell lung cancer.

Currently, particular focus is placed on the implication of autophagy in a variety of human diseases, including cancer. Discovery of small-molecule modulators of autophagy as well as their potential use as anti-cancer therapeutic agents would be of great significance. To this end, a series of curcumin analogs previously synthesized in our laboratory were screened. Among these compounds, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one (CA-5f) was identified as a potent late-stage macroautophagy/autophagy inhibitor via inhibiting autophagosome-lysosome fusion. We found that CA-5f neither impaired the hydrolytic function nor the quantity of lysosomes. Use of an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic screen in combination with bioinformatics analysis suggested that treatment of human umbilical vein endothelial cells (HUVECs) with CA-5f for 1 h suppressed the levels of cytoskeletal proteins and membrane traffic proteins. Subsequent studies showed that CA-5f exhibited strong cytotoxicity against A549 non-small cell lung cancer (NSCLC) cells, but low cytotoxicity to normal human umbilical vein endothelial cells (HUVECs), by increasing mitochondrial-derived reactive oxygen species (ROS) production. Moreover, CA-5f effectively suppressed the growth of A549 lung cancer xenograft as a single agent with an excellent tolerance in vivo. Results from western blot, immunofluorescence, and TdT-mediated dUTP nick end labeling (TUNEL) assays showed that CA-5f inhibited autophagic flux, induced apoptosis, and did not affect the level of CTSB (cathepsin B) and CTSD (cathepsin D) in vivo, which were consistent with the in vitro data. Collectively, these results demonstrated that CA-5f is a novel late-stage autophagy inhibitor with potential clinical application for NSCLC therapy. Abbreviations: 3-MA, 3-methyladenine; ANXA5, annexin A5; ATG, autophagy related; CA-5f, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; DMSO, dimethyl sulfoxide; DNM2, dynamin 2; EBSS, Earle's balanced salt solution; GFP, green fluorescent protein; HCQ, hydroxyl CQ; HEK293, human embryonic kidney 293; HUVEC, human umbilical vein endothelial cells; LAMP1, lysosomal associated membrane protein 1; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LDH, lactic acid dehydrogenase; LMO7, LIM domain 7; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; NAC, N-acetyl cysteine; MYO1E, myosin IE; NSCLC, non-small cell lung cancer; PARP1, poly(ADP-ribose) polymerase 1; PI, propidium iodide; RFP, red fluorescent protein; ROS, reactive oxygen species; SQSTM1, sequestosome 1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

4,4'-(Propane-1,3-di-yl)dipyridinium tetra-chloridonickelate(II).

The title compound, (C(13)H(16)N(2))[NiCl(4)] or (H(2)bpp)·NiCl(4) [bpp is 1,3-bis-(4-pyrid-yl)propane], is isostructural with its already reported Cu, Zn and Hg analogues. The structure consists of a doubly charged (H(2)bpp)(2+) cation and a tetra-hedral [NiCl(4)](2-) dianion. Both pyridyl N atoms are protonated and form a (H(2)bpp)(2+) cation which adopts an anti-anti conformation with a dihedral angle of 6.287 (7)° between the pyridyl rings. The two pyridyl N atoms are both involved in strong N-H⋯Cl hydrogen bonds, which link both units into a dimer.

[An experimental study on the influence of osteoporosis to bone repairing with bone matrix gelatin in ovariectomized rats].

OBJECTIVE: To study the influence of experimentally osteoporosis to osteogenic efficiency of bone matrix gelatin(BMG) implanted into the calvarial defects of rats. METHODS: Sixty-eight female SD rats of 12 +/- 1 weeks were randomly divided into two groups with 34 rats in each group. The ovaries were excised in the ovariectomized group (VG). The control group underwent sham surgery. Ninety days after ovariectomy, 10 rats from each group were examined to ensure the formation of postmenopausal osteoporosis by measuring bone density of the femur with single photon absorptiometric measurements. A critical-sized (8 mm in diameter) calvarial defect was created on the rest of 48 rats. Bone matrix gelatin was implanted to the defect. The rats were scarified at the 21st and 56th day after surgery respectively. The new bone forming capability of BMG was evaluated with undecalcified histological observation, tetracycline fluorescence marker, quantitative bone histomorphometry, At 90th day after ovariectomy, bone density of scanning electron microscopy and X-ray spectrometry. RESULTS: OVG showed very significant difference compared with the control group (0.315 +/-.015) g/cm2 vs [(0.347 +/- 0.017) g/cm2, P < 0.01 ]. At the 21st day following the implantation operation, new bone formed within the bone defects in both groups. The amount of new bone in OVG was lower than the control group. The tetracycline-labeled region in the bone defect was sparser in the OVG. At the 56th day, the bone defects healed mostly in the control group but fibrous tissue filled parts of bone defect in the OVG. The distance between two fluorescent lines of incorporated tetracycline and the mean mineralization deposition were significantly lower in the OVG than the control at the 21st day and 56th day. Mineralization of callus in OVG was inferior. Significant difference was found between the OVG and the control group in the calcium to phosphate ratio of callus in bone defects at the two time-points. CONCLUSIONS: Experimentally induced osteoporosis depressed osteogenic efficiency of BMG, suggesting that estrogen could play an important role in bone remodeling with bone substitute participating.

[Effects of stilbestrol on bone growth and metabolism in ovariectomized rats].

OBJECTIVE: To study the effects of stilbestrol on bone growth and metabolism in ovariectomized rats. METHODS: Twenty-seven 3-month-old female SD rats were randomly divided into 3 equal groups, namely the control group, ovariectomized group (OEG), and OEG+estrogen (OEG+E) group. After ovariectomy, the rats in the third group were given stilbestrol 0.022 5 mg/d x kg x b x w by intragastric gavage for 90 d. A digital image analysis system was used for the measurement of histomorphometric parameters in the proximal tibial metaphysis of the rats. The three-dimensional structure of the femur was observed with scanning electron microscope, with the blood cholesterol level determined and the main viscera weighted. RESULTS: In comparison with the control rats, the trabecular bone area (TbAr) of ovariectomized rats was significantly reduced (-65%), trabecular bone space (TbSp) enlarged (+189%), and the bone formation rate (BFR) accelerated (+91%). The cancellous bone of the femurs manifested obvious signs of thinning, roughening and resorption lacunae to cause trabecular bone discontinuance, and osteoporosis occurred. The rats treated with stilbestrol, in contrast, had increased TbAr (by 128%), reduced TbSp (by 63%), lowered BFR (by 49%), and reduced osteoclasts (by 32%). The cancellous bone of the femurs was well arranged in close connection with each other, and the high cholesterol level and endocrinic changes in response to ovariectomy was corrected by stilbestrol, which also caused the increase of the uterine weight. CONCLUSION: Stilbestrol may help prevent primary osteoporosis but may increase the risk of female genital cancer.