Control of arbuscule development by a transcriptional negative feedback loop in Medicago.

Most terrestrial plants establish a symbiosis with arbuscular mycorrhizal fungi (AMF), which provide them with lipids and sugars in exchange for phosphorus and nitrogen. Nutrient exchange must be dynamically controlled to maintain a mutually beneficial relationship between the two symbiotic partners. The WRI5a and its homologues play a conserved role in lipid supply to AMF. Here, we demonstrate that the AP2/ERF transcription factor MtERM1 binds directly to AW-box and AW-box-like cis-elements in the promoters of MtSTR2 and MtSTR, which are required for host lipid efflux and arbuscule development. The EAR domain-containing transcription factor MtERF12 is also directly activated by MtERM1/MtWRI5a to negatively regulate arbuscule development, and the TOPLESS co-repressor is further recruited by MtERF12 through EAR motif to oppose MtERM1/MtWRI5a function, thereby suppressing arbuscule development. We therefore reveal an ERM1/WRI5a-ERF12-TOPLESS negative feedback loop that enables plants to flexibly control nutrient exchange and ensure a mutually beneficial symbiosis.

Microalgal flocculation and sedimentation: spatiotemporal evaluation of the effects of the pH and calcium concentration.

The high cost of harvesting microalgae is a major hurdle for the microalgae industry, and an efficient pre-concentration method is required. In this study, the effects of using different pH values (between pH 3 and 11) and calcium (Ca2+) concentrations (between 0 and 5 mM) on Chlorella vulgaris sedimentation were investigated by evaluating the spacio-temporal distributions of microalgae cells. Fast and efficient sedimentation occurred (within 10 min) at a high Ca2+ concentration (5 mM) at pH 9 and 11. However, the sediment volume was lower at a Ca2+ concentration of 3 mM than at a Ca2+ concentration of 5 mM. This indicated that the Ca2+ concentration strongly affected the sediment volume. Fast sedimentation and a low sediment volume were found at pH 7 and a Ca2+ concentration of 5 mM, probably because of the neutral charge in the system (adhesion to calcium precipitates would have occurred at a high pH). The highest Ca2+ recovery (82%) was achieved when sediment produced at pH 11 and a Ca2+ concentration of 5 mM was acidified to pH 3.

Oriented Growth of Thin Films of Covalent Organic Frameworks with Large Single-Crystalline Domains on the Water Surface.

It has been a longstanding challenge to rationally synthesize thin films of organic two-dimensional (2D) crystals with large single-crystalline domains. Here, we present a general strategy for the creation of 2D crystals of covalent organic frameworks (COFs) on the water surface, assisted by a charged polymer. The morphology of the preorganized monomers underneath the charged polymer on the water surface and their diffusion were crucial for the formation of the organic 2D crystals. Thin films of 2D COFs with an average single-crystalline domain size of around 3.57 ± 2.57 µm2 have been achieved, and their lattice structure, molecular structure, and grain boundaries were identified with a resolution down to 3 Å. The swing of chain segments and lattice distortion were revealed as key factors in compensating for the misorientation between adjacent grains and facilitating error corrections at the grain boundaries, giving rise to larger single-crystalline domains. The generality of the synthesis method was further proved with three additional 2D COFs. The oriented single-crystalline domains and clear grain boundaries render the films as model materials to study the dependence of the vertical conductivity of organic 2D crystals on domain sizes and chemical structures, and significant grain boundary effects were illustrated. This study presents a breakthrough in the controlled synthesis of organic 2D crystals with structural control at the molecular level. We envisage that this work will inspire further investigation into the microstructure-intrinsic property correlation of 2D COFs and boost their application in electronics.

PCL/Collagen/UA Composite Biomedical Dressing with Ordered Microfiberous Structure Fabricated by a 3D Near-Field Electrospinning Process.

In this work, a functionalized polycaprolactone (PCL) composite fiber combining calf-type I collagen (CO) and natural drug usnic acid (UA) was prepared, in which UA was used as an antibacterial agent. Through 3D near-field electrospinning, the mixed solution was prepared into PCL/CO/UA composite fibers (PCUCF), which has a well-defined perfect arrangement structure. The influence of electrospinning process parameters on fiber diameter was investigated, the optimal electrospinning parameters were determined, and the electric field simulation was conducted to verify the optimal parameters. The addition of 20% collagen made the composite fiber have good hydrophilicity and water absorption property. In the presence of PCUCF, 1% UA content significantly inhibited the growth rate of Gram-positive and negative bacteria in the plate culture. The AC-PCUCF (after crosslinking PCUCF) prepared by crosslinking collagen with genipin showed stronger mechanical properties, water absorption property, thermal stability, and drug release performance. Cell proliferation experiments showed that PCUCF and AC-PCUCF had no cytotoxicity and could promote cell proliferation and adhesion. The results show that PCL/CO/UA composite fiber has potential application prospects in biomedical dressing.

Preparation of Coralline Hydroxyapatite Implant with Recombinant Human Bone Morphogenetic Protein-2-Loaded Chitosan Nanospheres and Its Osteogenic Efficacy.

OBJECTIVE: Spinal fusion is one of the most common surgical interventions for spine reconstruction. Despite the efforts to promote osteogenesis after spinal fusion, osteogenesis after spinal fusion remains a clinical challenge and new methods are still needed. The bone morphogenetic protein-2 (BMP-2) is a widely reported factor that can facilitate the osteogenesis in spinal fusion. In previous research, we found that the delivery of chitosan nanospheres could promote the effects of BMP-2 on osteogenic activity. The coralline hydroxyapatite (CHA) is one of the most frequently used implants in bone fusion. However, up to now no study has focused on the osteogenic efficacy of the CHA composite with recombinant human BMP-2 (rhBMP-2)-loaded chitosan nanospheres. This study aimed to investigate the effects of the CHA implant with rhBMP-2-loaded chitosan nanospheres on osteogenesis in spinal fusion. METHODS: The rhBMP-2-loaded microspheres and CHA composite (rhBMP-2 microspheres/CHA) were prepared and were used for implantation of the rats. All SD rats were divided into four groups: the rhBMP-2 microspheres/CHA composite group (containing 0.5 mg rhBMP-2), the rhBMP-2-loaded CHA (rhBMP-2/CHA) composite group (containing 0.5 mg rhBMP-2), the blank CHA group, and the negative control group. The microsphere morphology was scanned and analyzed using a scanning electron microscope. Micro-computed tomography examination and three-dimensional reconstruction were performed 4 weeks after the surgery. Hematoxylin and eosin staining was conducted for histological analysis. Both alkaline phosphatase (ALP) and calcium content were measured. RESULTS: The rhBMP-2-loaded CHA (rhBMP-2/CHA) composite was successfully prepared. Spherical regularity and a smooth and unwrinkled surface of the spheres were observed in all chitosan (CS)/rhBMP-2 microspheres. No side effects, infections, or abnormal behaviors were found in the animals. After 4 weeks of surgery, obvious new bone formation and bone fusion could be observed around the implant in both the rhBMP-2 microspheres/CHA composite group and the rhBMP-2/CHA composite group. No ectopic osteogenesis was found in the vertebral canal or other muscle tissues. After 4 weeks of implantation, in both the rhBMP-2 microspheres/CHA composite group and the rhBMP-2/CHA composite group, osteoid tissues could be found, and bone cells, bone marrow, and trabecular bone turned into mature sclerotin, obvious bone tissue formation could be also seen. Both ALP activity and calcium content in the rhBMP-2 microspheres/CHA composite group (6.52 ± 0.50 kat/g and 17.54 ± 2.49 µg/mg) were significantly higher than in all other groups. CONCLUSION: The composite with rhBMP-2-loaded CS nanospheres could enhance osteogenic efficacy and increase the ALP activity and calcium content. These results might provide a novel method for osteogenesis in spinal fusion and offer new insight into the role of BMP-2 in osteogenesis.

Effect of recombinant human bone morphogenetic protein delivered by chitosan microspheres on ectopic osteogenesis in rats.

In the present study, the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered by chitosan (CS) microspheres on ectopic osteogenesis was investigated in a rat model. rhBMP-2-loaded CS microspheres and blank CS microspheres were prepared. A total of 24 male Sprague Dawley rats were divided into 4 groups with 6 rats in each group: The CS/rhBMP-2 group, the rhBMP-2 group, in which rhBMP-2 was directly implanted (rhBMP-2 dose in either group, 1 mg), the CS blank group and the control group. X-ray was performed at 4 weeks after ectopic osteogenesis surgery and micro-computed tomography (CT) examination was scheduled at 1, 2, 3 and 4 weeks after the surgery to determine ectopic osteogenesis in the different groups. Histological analysis, and determination of alkaline phosphatase (ALP) activity and calcium content were also performed. The mean diameter of the osteoid tissues was 1.1±0.3 cm (range, 0.8-1.4 cm) in the CS/rhBMP-2 group, which was significantly bigger than that in the rhBMP-2 group (0.3±0.1 cm; range, 0.1-0.4 cm) at 4 weeks after the surgery. X-ray analysis and micro-CT scan indicated that the area of high-density tissues and the radionuclide intensity, as well as bone volume in the 3-dimensional reconstruction were greatest in the CS/rhBMP-2 group, followed by those in the rhBMP-2 group. All parameters, including bone mineral density, tissue mineral density, tissue mineral content and bone volume fraction, were significantly higher in the CS/rhBMP-2 group at 3 and 4 weeks after the surgery, compared with those in the rhBMP-2 group. The histological analysis, ALP activity analysis and determination of calcium content revealed that the CS/rhBMP-2 system had the greatest ability to induce osteoblast differentiation. In conclusion, the CS/rhBMP-2 microsphere delivery system significantly enhanced the induction and promotion effects of rhBMP-2 regarding ectopic osteogenesis. The present study enhances the basic data available for future application of the CS/rhBMP-2 microspheres delivery system and provides a deeper understanding of the role of BMP-2 in bone regeneration.

Integrating 3D-printed PHBV/Calcium sulfate hemihydrate scaffold and chitosan hydrogel for enhanced osteogenic property.

We developed the 3D-printed poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/calcium sulfate hemihydrate (PHBV/CaSH) scaffolds by using fused deposition modelling (FDM) technique and then coated the scaffolds with chitosan (CS) acetic acid solution. After drying and neutralization, CS hydrogel was formed on the surface of the scaffolds. The resultant PHBV/CaSH/CS scaffolds could promote the adhesion and proliferation of rat bone marrow stromal cells (rBMSCs) and enhance the osteogenesis of rBMSCs by up-regulating the expression level of osteogenic genes compared to the PHBV and PHBV/CaSH scaffolds. In vivo studies further demonstrated the PHBV/CaSH/CS scaffolds could effectively promote new bone formation. Therefore, integrating 3D-printed PHBV/CaSH scaffold and CS hrydogel represents a novel strategy to promote osteogensis property, showing full potential for bone defects repair.

Efficient delivery of recombinant human bone morphogenetic protein (rhBMP-2) with dextran sulfate-chitosan microspheres.

Bone morphogenetic protein-2 (BMP-2) serves an important role in the development of bone and cartilage. However, administration of BMP-2 protein alone by intravenous delivery is not very effective. Sustained delivery of stabilized BMP-2 by carriers has been proven necessary to improve the osteogenesis effect of BMP-2. The present study constructed a novel drug delivery system using dextran sulfate (DS)-chitosan (CS) microspheres and investigated the efficiency of the delivery system on recombinant human bone morphogenetic protein (rhBMP-2). The microsphere morphology, optimal ratio of DS/CS/rhBMP-2, and drug loading rate and entrapment efficiency of rhBMP-2 CS nanoparticles were determined. L929 cells were used to evaluate the cytotoxicity and effect of DS/CS/rhBMP-2 microspheres on cell proliferation. Differentiation study was conducted using bone marrow mesenchymal stem cells (BMSCs-C57) cells treated with DS/CS/rhBMP-2 microspheres or the control microspheres. The DS/CS/rhBMP-2 microspheres delivery system was successfully established. Subsequent complexation of rhBMP-2-bound DS with polycations afforded well defined microspheres with a diameter of ~250 nm. High protein entrapment efficiency (85.6%) and loading ratio (47.245) µg/mg were achieved. Release of rhBMP-2 from resultant microspheres persisted for over 20 days as determined by ELISA assay. The bioactivity of rhBMP-2 encapsulated in the CS/DS microsphere was observed to be well preserved as evidenced by the alkaline phosphatase activity assay and calcium nodule formation of BMSCs-C57 incubated with rhBMP-2-loaded microspheres. The results demonstrated that microspheres based on CS-DS polyion complexes were a highly efficient vehicle for delivery of rhBMP-2 protein. The present study may provide novel orientation for bone tissue engineering for repairing and regenerating bone defects.

Bioinspired interconnected hydrogel capsules for enhanced catalysis.

Polysaccharide-based hydrogel capsules with interconnected inner membranes have been prepared. The obtained capsules have a multi-layer internal structure composed of many invaginations of interconnected hydrogel membranes as mimics of the cristae in mitochondria. These cristae-like internal membranes endow the capsules with high specific area, which makes the capsules ideal supporting materials for catalytic species such as metal nanoparticles and enzymes.

[Radiological evaluation of dextran sulfate/recombinant human bone morphogenetic protein 2/chitosan composite microspheres combined with coral hydroxyapatite artificial bone in repairing large segmental bone defects].

Objective: To evaluate the osteogenic effect of dextran sulfate/recombinant human bone morphogenetic protein 2/chitosan (DS/rhBMP-2/CS) combined with coralline hydroxyapatite (CHA) in repairing large segmental bone defects by radiographic feature. Methods: Fifty-seven 24-week-old male New Zealand rabbits were prepared for establishing right radius bone defect model of 20 mm in length. In which 54 rabbits were randomly divided into 3 groups ( n=18), and the CHA, DS/rhBMP-2/CS/CHA, and rhBMP-2/CHA artificial bone grafts were implanted into the bone defect in groups A, B, and C respectively; the remaining 3 rabbits were implanted nothing as blank control group. After operation, the gross condition of the animals was observed; at 4, 8, and 12 weeks after operation, X-ray film observation, Micro-CT scanning, and three-dimensional reconstruction were performed to obtain the volume of the new bone. Results: The experimental animals recovered well and were in normal condition. X-ray observation showed that the bone healing in group B was better than that in groups A and C at each time point. At each time point after operation, the X-ray scores of group B were significantly higher than that of group A and group C ( P<0.05); the scores of group C at 8 and 12 weeks after operation were significantly higher than that of group B ( P<0.05). Micro-CT scanning and three-dimensional reconstruction observation showed that at each time point after operation in group A, the bone defect area had less bone formation and poor osteogenesis; in group B, there were many new bone tissues in bone defect area, and the bone remodeling was well, and gradually closed to normal bone morphology at 12 weeks; in group C, there were many new bone tissues in bone defect area, but the bone formation was general. The new bone volume of group B was significantly higher than that of group A and group C ( P<0.05) at each time point after operation, and the score of group C was higher than that of group A at 8 weeks after operation ( P<0.05). Conclusion: The osteogenic effect of DS/rhBMP-2/CS/CHA sustained-release artificial bone is much better than that of single CHA and rhBMP-2/CHA, which can provide a new idea for treating bone defect by using bone tissue engineering in the future.

[ECTOPIC OSTEOGENESIS EVALUATION OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 LOADED CHITOSAN/DEXTRAN SULFATE BY MICRO-CT].

UNLABELLED: OBJECTIVE To evaluate the ectopic osteogenesis of recombinant human bone morphogenetic protein 2 (rhBMP-2) loaded chitosan (CS)/dextran sulfate (DS) by micro-CT. METHODS: rhBMP-2/CS/DS microspheres were prepared by the ionic crosslinking and its shape was observed under the scanning electron microscope. The release of rhBMP-2 was determined from resultant microspheres by ELISA assay. Forty-eight Sprague Dawley male rats were randomly divided into 4 groups (n = 12), quadriceps muscle bag model was made, gelatin sponge (group A), CS/DS microspheres (group B), rhBMP-2 (group C), and CS/DS/rhBMP-2 microspheres (group D) were implanted into the bags respectively. The tissue samples with heterotopic ossification were harvested for micro-CT scanning at 4, 8, 12, and 16 weeks. The tissue mineral density (TMD), bone volume fraction (BVF), trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and tissue mineral content (TMC) were measured. RESULTS: The prepared rhBMP-2/CS/DS microspheres with smooth surfaces were spherical and evenly disperses without obvious agglomeration. At 2 hours, microsphere started a sudden release period in vitro; the release reached a peak at 2 days; and the release cycle lasted about 20 days. The rats survived to the end of the experiment. At each time point after operation, no radiation developed and no osteogenesis was observed by three dimensional reconstruction in groups A and B. However, radioactive strength and reconstructed bone tissue gradually increased in groups C and D, and group D had more radioautography and more bone tissues than group C. At each time point, TMD, BVF, Tb.Th, Tb.N, BMD, and TMC of groups A and B were zero. Ectopic bone formed with time, the other parameters showed an increasing trend except Tb.N in groups C and D, showing significant difference when compared with groups A and B at each time point (P < 0.05). There was no significant difference between groups C and D at 4 weeks (P > 0.05); the parameters of group D were significantly higher than those of group C at 8-16 weeks (P < 0.05). CONCLUSION: rhBMP-2/CS/DS microspheres have stronger ability of ectopic bone formation than single rhBMP-2.

[Computer aid designed operation of complicated calcaneal fracture by 3D simulation].

OBJECTIVE: To precisely treat complicated calcaneal fracture by 3D simulation through computer aid designed operation. METHODS: From November 2007 to March 2008, 38 patients of calcaneal fracture were treated. There were 29 males and 9 females aged 14-69 years old (average 29.8 years old). According to Sanders classification, there were 4 patients of type I, 14 of type II, 12 of type III, and 8 of type IV. The time between injury and surgery was 3 hours to 5 days. The CT images of calcaneal fracture of 38 patients were put into computer for 3D reconstruction, then the Böhler angles were measured and bone grafting angles were designed. According to the angle surveyed by the computer, the individual-oriented operation program was made, and then the operation was done under C-arm X-ray machine. RESULTS: The preoperative Böhler angel was (34.58 +/- 4.38) degrees in the normal side and (8.33 +/- 12.62) degrees in the injured side, indicating there was significant difference (P < 0.05). During the process of the poking reduction by 3D simulation, when the bone rotating angle was (28.84 +/- 6.51) degrees, the Böhler angel was restored to (32.86 +/- 1.72) degrees, indicating there was no significant difference when compared with the normal side before operation (P > 0.05), and significant difference compared with the injured side before operation (P < 0.05). Twenty-eight patients were followed up for 12-22 months (average 18 months). The Böhler angel was restored to (32.41 +/- 1.42) degrees 1 year after operation. According to the foot function scoring system made by American Ankle Surgery Association, 16 cases were graded as excellent, 10 as good, 1 as fair, 1 as poor, and the excellent and good rate was 92.9%. CONCLUSION: Computer aid designed operation of complicated calcaneal fracture by 3D simulation technique can restore the Böhler angel and subtalar joint precisely. It is an effective supplementary treatment method for calcaneal fracture.