Deciphering molecular heterogeneity and dynamics of human hippocampal neural stem cells at different ages and injury states.

While accumulated publications support the existence of neurogenesis in the adult human hippocampus, the homeostasis and developmental potentials of neural stem cells (NSCs) under different contexts remain unclear. Based on our generated single-nucleus atlas of the human hippocampus across neonatal, adult, aging, and injury, we dissected the molecular heterogeneity and transcriptional dynamics of human hippocampal NSCs under different contexts. We further identified new specific neurogenic lineage markers that overcome the lack of specificity found in some well-known markers. Based on developmental trajectory and molecular signatures, we found that a subset of NSCs exhibit quiescent properties after birth, and most NSCs become deep quiescence during aging. Furthermore, certain deep quiescent NSCs are reactivated following stroke injury. Together, our findings provide valuable insights into the development, aging, and reactivation of the human hippocampal NSCs, and help to explain why adult hippocampal neurogenesis is infrequently observed in humans.

Incidence and risk factors of recurrence in limb osteomyelitis patients after antibiotic-loaded cement spacer for definitive bone defect treatment.

Aims: This study was designed to characterize the recurrence incidence and risk factors of antibiotic-loaded cement spacer (ALCS) for definitive bone defect treatment in limb osteomyelitis. Methods: We included adult patients with limb osteomyelitis who received debridement and ALCS insertion into the bone defect as definitive management between 2013 and 2020 in our clinical centre. The follow-up time was at least two years. Data on patients' demographics, clinical characteristics, and infection recurrence were retrospectively collected and analyzed. Results: In total, 314 patients with a mean age of 52.1 years (SD 12.1) were enrolled. After a mean of 50 months' (24 to 96) follow-up, 53 (16.9%) patients had infection recurrence including 32 tibiae, ten femora, ten calcanea, and one humerus. Of all patients with recurrence, 30 (9.6%) occurred within one year and 39 (12.4%) within two years. Among them, 41 patients needed reoperation, five received antibiotics treatment only, and seven ultimately required amputations. Following multivariable analysis, we found that patients infected with Gram-negative bacilli were more likely to have a recurrence (odds ratio (OR) 2.38, 95% confidence interval (CI) 1.20 to 6.94; p = 0.046) compared to Staphylococcus aureus; segmental bone defects (OR 5.25, 95% CI 1.80 to 15.26; p = 0.002) and smoking (OR 3.00, 95% CI 1.39 to 6.50; p = 0.005) were also independent risk factors for recurrence after treatment. Conclusion: Permanent ALCS might be an alternative strategy for definitive bone defect management in selected osteomyelitis cases. However, the overall high recurrence found suggests that it should be cautiously treated. Additionally, segmental defects, Gram-negative infections, and smoking were associated with an increased risk of infection recurrence.

Biomimetic Antithrombotic Tissue-Engineered Vascular Grafts for Converting Cholesterol and Free Radicals into Nitric Oxide.

Small-diameter tissue-engineered vascular grafts (sdTEVGs) are essential materials used in bypass or replacement surgery for cardiovascular diseases; however, their application efficacy is limited because of patency rates, especially under hyperlipidemia, which is also clinically observed in patients with cardiovascular diseases. In such cases, improving sdTEVG patency is challenging because cholesterol crystals easily cause thrombosis and impede endothelialization. Herein, the development of a biomimetic antithrombotic sdTEVG incorporating cholesterol oxidase and arginine into biomineralized collagen-gold hydrogels on a sdTEVG surface is described. Biomimetic antithrombotic sdTEVGs represent a multifunctional substrate for the green utilization of hazardous substances and can convert cholesterol into hydrogen peroxide, which can react with arginine to generate nitric oxide (NO). NO is a vasodilator that can simulate the antithrombotic action of endothelial cells under hyperlipidemic conditions. In vivo studies show that sdTEVGs can rapidly produce large amounts of NO via a cholesterol catalytic cascade to inhibit platelet aggregation, thereby improving the blood flow velocity and patency rates 60 days after sdTEVG transplantation. A practical and reliable strategy for transforming "harmful" substances into "beneficial" factors at early transplantation stages is presented, which can also promote vascular transplantation in patients with hyperlipidemia.

Surface-Engineered Ti3 C2 Tx with Tunable Work Functions for Highly Efficient Polymer Solar Cells.

Ti3 C2 Tx , as a newly investigated 2D material, has gained great attention owing to its metallic conductivity, tunable work function (WF ), and unique electrical property. However, its WF can be further adjusted to meet the needs of optoelectronic devices. Here, surface-engineered Ti3 C2 Tx is fabricated with tunable WF by treating with ethanolamine and rhodium chloride (RhCl3 ). Ethanolamine treated Ti3 C2 Tx can induce the chemical adsorption of NH2 on Ti3 C2 Tx with hydrogen-bonding, which causes the decreased WF , while chemical doping with RhCl3 leads to the improvement of WF , which is achieved by the downshift of Femi level of Ti3 C2 Tx . Moreover, the ethanolamine and RhCl3 can effectively passivate the vacancies of Ti. As such, the surface-engineered Ti3 C2 Tx is more suitable as buffer layer for polymer solar cells (PSCs) by enhancing the interfacing characteristics of the Ti3 C2 Tx /active layer. The PSCs with engineered Ti3 C2 Tx for electron or hole transport layers can exhibit a power conversion efficiency of 15.88% or 15.54%. These efficiencies can be compared with those of devices with a conventional transport layer. This work provides a facile strategy to realize the work function tunability of Ti3 C2 Tx , and also shows that the tuned Ti3 C2 Tx has a certain application prospect in photovoltaic devices.

High-efficiency ternary polymer solar cells with optimized morphology of active layers enabled by few-layered ß-InSe nanosheets.

Due to the excellent electrical and optical properties, few-layered ß-indium selenide (InSe) nanosheets are successfully introduced into the active layer of polymer solar cells (PSCs) as the third component for the first time. The addition of few-layered ß-InSe nanosheets optimizes the absorption, crystallinity and vertical component distribution of the active layer. Compared with the binary devices, the ternary devices exhibit optimized bulk morphology and reduced charge recombination. The power conversion efficiency (PCE) of PSCs based on the PM6 : Y6 system is obviously improved from 15.02% to 16.56% due to the increasing short-circuit current and fill factor. The mechanism accounting for the morphological change in the ternary active layer is investigated in depth. Moreover, the efficacy of ß-InSe in long-term stability and other active layer systems of PSCs is confirmed. Therefore, this work demonstrates that few-layered ß-InSe has bright prospects in photovoltaic devices.

The species evenness of "prey" bacteria correlated with Bdellovibrio-and-like-organisms (BALOs) in the microbial network supports the biomass of BALOs in a paddy soil.

To seek how soil biotic and abiotic factors which might shape the Bdellovibrio-and-like-organisms community, we sampled paddy soils under different fertilization treatments including fertilization without nitrogen (Control), the nitrogen use treatment (N) and the nitrogen overuse one (HNK) at three rice growing stages. The abundances of BALOs were impacted by the rice-growing stages but not the fertilization treatments. The abundances of Bdellovibrionaceae-like were positively associated with soil moisture, which showed a negative relationship with Bacteriovoracaceae-like bacteria. High-throughput sequencing analysis of the whole bacterial community revealed that the α-diversity of BALOs was not correlated with any soil properties data. Network analysis detected eight families directly linked to BALOs, namely, Pseudomonadaceae, Peptostreptococcaceae, Flavobacteriaceae, Sediment-4, Verrucomicrobiaceae, OM27, Solirubrobacteraceae and Roseiflexaceae. The richness and composition of OTUs in the eight families were correlated with different soil properties, while the evenness of them had a positive effect on the predicted BALO biomass. These results highlighted that the bottom-up control of BALOs in paddy soil at least partially relied on the changes of soil water content and the diversity of bacteria directly linked to BALOs in the microbial network.

The limited effects of carbonaceous material amendments on nitrite-oxidizing bacteria in an Alfisol.

Carbonaceous materials are soil conditioners that affect nitrogen cycles. However, how carbonaceous materials influence nitrite-oxidizing bacteria (NOB) is yet unclear. In this study, we investigated the NOB community and its potential activities under different treatments (control, biochar, straw, limestone, biochar + limestone, and straw + limestone) in an Alfisol, a type of arable soil depleted in calcium carbonate but enriched in aluminum- and iron-bearing minerals. Treatments with limestone increased soil pH, and straw inputs caused an increment of available potassium (AK). Ammonia (NH4+) was inversely changed under the straw and biochar + limestone amendments. None of the treatments significantly impacted the abundance of Nitrobacter (nxrA) or the potential nitrite oxidation activity (PNO). The abundance of Nitrospira (nxrB) increased in the biochar + limestone-treated samples and was significantly correlated with PNO, pH, and AK. High-throughput sequencing results showed that the α-diversity of NOB did not change in response to the treatments. The dominant Nitrobacter OTUs were affiliated within the Clusters 3, 4, 8, and 9 (a new cluster named in this study), while those of Nitrospira were in the lineage II and Namibian soil cluster 2. The limited compositional variation for Nitrobacter was explained by pH, and that for Nitrospira by pH, TN, and NH4+. Among all available data in this study, the richness of Nitrospira was the most important predictor (73%) for PNO. Therefore, we assumed that the community of nitrite oxidizers (Nitrospira) could be relatively redundant in function, supported by the observation that the carbonaceous inputs did not impact either the potential activity or the α-diversity but did affect the abundance and community composition.

Solution-Processable PEDOT:PSS:α-In2Se3 with Enhanced Conductivity as a Hole Transport Layer for High-Performance Polymer Solar Cells.

Two-dimensional (2D) nanosheets have attracted significant attention in photovoltaic devices in recent years owing to their outstanding photoelectric properties. Herein, 2D α-In2Se3 nanosheets with high conductivity and suitable work function are synthesized by liquid-phase exfoliation method. To ameliorate the low conductivity of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) (2.21 × 10-3 S cm-1), α-In2Se3 nanosheets are directly added into PEDOT:PSS to obtain the PEDOT:PSS:α-In2Se3 composite film. The composite film exhibits excellent optical transmittance, suitable work function, and enhanced conductivity (1.54 × 10-2 S cm-1). To profoundly investigate the mechanism of conductivity improvement, X-ray photoelectron spectroscopy, Raman spectroscopy, electron paramagnetic resonance, and atomic force microscopy are conducted. The results show that the synergistic effect of 2D α-In2Se3 nanosheets and isopropyl alcohol/deionized water cosolvent screens the Coulombic attraction among PEDOT and PSS. The screening effect results in the partial removal of PSS and the benzoid-quinoid transition of PEDOT. In addition, α-In2Se3 nanosheets may serve as physical linkers for PEDOT chains. Both these effects are beneficial to increase the interfacial contact area between PEDOT chains and form a larger conductive network of PEDOT, leading to an enhanced conductivity. The composite film is first employed as a hole transport layer (HTL) in polymer solar cells (PSCs). The power conversion efficiency (PCE) of the poly[2,6-(4,8-bis(5-(2-ethylhexyl)thiophen-2-yl)benzo[1,2-b:4,5-b']dithiophene)-co-(1,3-di(5-thiophene-2-yl)-5,7-bis(2-ethylhexyl)benzo[1,2-c:4,5-c']dithiophene-4,8-dione)] (PBDB-T):3,9-bis(2-methylene(3-(1,1-dicyanomethylene)-indanone))-5,5,11,11-tetrakis(4-hexylphenyl)dithieno-[2,3-d:2',3'-d']-s-indaceno[1,2-b:5,6-b']dithiophene (ITIC)-based device with composite HTL is 10% higher than that of the unmodified PBDB-T:ITIC-based device, and the maximum PCE of 15.89% is achieved in the (poly[(2,6-(4,8-bis(5-(2-ethylhexyl)-4-fluorothiophen-2-yl)benzo[1,2-b:4,5-b']dithiophene))-co-(1,3-di(5-thiophene-2-yl)-5,7-bis(2-ethylhexyl)-benzo[1,2-c:4,5-c']dithiophene-4,8-dione))] (PM6):(2,2'-((2Z,2Z)-((12,13-bis(2-ethylhexyl)-3,9-diundecyl-12,13-dihydro-[1,2,5]thiadiazolo[3,4-e]thieno[2,″3″:4',50]thieno[2',3':4,5]pyrrolo[3,2-g]thieno[2',3':4,5]thieno[3,2-b]indole-2,10-diyl)bis(methanylylidene))bis(5,6-difluoro-3-oxo-2,3-dihydro-1H-indene-2,1-diylidene))dimalononitrile) (Y6) system. More interestingly, the stability of devices with composite HTL is improved owing to the partial removal of PSS. Thus, the PEDOT:PSS:α-In2Se3 composite can be a potential HTL material in PSCs.

[Spatial-temporal Characteristics and Driving Factors of Greenhouse Gas Emissions from Rivers in a Rapidly Urbanizing Area].

Rivers play an important role in greenhouse gas emissions. Over the past decade, because of global urbanization trends, rapid land use changes have led to changes in river ecosystems that have had a stimulating effect on the greenhouse gas production and emissions. Presently, there is an urgent need for assessments of the greenhouse gas concentrations and emissions in watersheds. Therefore, this study was designed to evaluate river-based greenhouse gas emissions and their spatial-temporal features as well as possible impact factors in a rapidly urbanizing area. The specific objectives were to investigate how river greenhouse gas concentrations and emission fluxes are responding to urbanization in the Liangtan River, which is not only the largest sub-basin but also the most polluted one in Chongqing City. The thin layer diffusion model method was used to monitor year-round concentrations of pCO2, CH4, and N2O in September and December 2014, and March and June 2015. The pCO2 range was (23.38±34.89)-(1395.33±55.45) Pa, and the concentration ranges of CH4 and N2O were (65.09±28.09)-(6021.36±94.36) nmol·L-1 and (29.47±5.16)-(510.28±18.34) nmol·L-1, respectively. The emission fluxes of CO2, CH4, and N2O, which were calculated based on the method of wind speed model estimations, were -6.1-786.9, 0.31-27.62, and 0.06-1.08 mmol·(m2·d)-1, respectively. Moreover, the CO2 and CH4 emissions displayed significant spatial differences, and these were roughly consistent with the pollution load gradient. The greenhouse gas concentrations and fluxes of trunk streams increased and then decreased from upstream to downstream, and the highest value was detected at the middle reaches where the urbanization rate is higher than in other areas and the river is seriously polluted. As for branches, the greenhouse gas concentrations and fluxes increased significantly from the upstream agricultural areas to the downstream urban areas. The CO2 fluxes followed a seasonal pattern, with the highest CO2 emission values observed in autumn, then successively winter, summer, and spring. The CH4 fluxes were the highest in spring and the lowest in summer, while N2O flux seasonal patterns were not significant. Because of the high carbon and nitrogen loads in the basin, the CO2 products and emissions were not restricted by biogenic elements, but levels were found to be related to important biological metabolic factors such as the water temperature, pH, DO, and chlorophyll a. The carbon, nitrogen, and phosphorus content of the water combined with sewage input influenced the CH4 products and emissions. Meanwhile, N2O production and emissions were mainly found to be driven by urban sewage discharge with high N2O concentrations. Rapid urbanization accelerated greenhouse gas emissions from the urban rivers, so that in the urban reaches, CO2/CH4 fluxes were twice those of the non-urban reaches, and all over the basin N2O fluxes were at a high level. These findings illustrate how river basin urbanization can change aquatic environments and aggravate allochthonous pollution inputs such as carbon, nitrogen, and phosphorus, which in turn can dramatically stimulate river-based greenhouse gas production and emissions; meanwhile, spatial and temporal differences in greenhouse gas emissions in rivers can lead to the formation of emission hotspots.

Quantitative Proteomics of Th-MYCN Transgenic Mice Reveals Aurora Kinase Inhibitor Altered Metabolic Pathways and Enhanced ACADM To Suppress Neuroblastoma Progression.

Neuroblastoma is a neural crest-derived embryonal tumor and accounts for about 15% of all cancer deaths in children. MYCN amplification is associated with aggressive and advanced stage of high-risk neuroblastoma, which remains difficult to treat and exhibits poor survival under current multimodality treatment. Here, we analyzed the transcriptomic profiles of neuroblastoma patients and showed that aurora kinases lead to poor survival and had positive correlation with MYCN amplification and high-risk disease. Further, pan-aurora kinase inhibitor (tozasertib) treatment not only induces cell-cycle arrest and suppresses cell proliferation, migration, and invasion ability in MYCN-amplified (MNA) neuroblastoma cell lines, but also inhibits tumor growth and prolongs animal survival in Th-MYCN transgenic mice. Moreover, we performed quantitative proteomics and identified 150 differentially expressed proteins after tozasertib treatment in the Th-MYCN mouse model. The functional and network-based enrichment revealed that tozasertib alters metabolic processes and identified a mitochondrial flavoenzyme in fatty acid ß-oxidation, ACADM, which is correlated with aurora kinases and neuroblastoma patient survival. Our findings indicate that the aurora kinase inhibitor could cause metabolic imbalance, possibly by disturbing carbohydrate and fatty acid metabolic pathways, and ACADM may be a potential target in MNA neuroblastoma.

A novel dressing seeded with embryonic artery CD133+ cells and loaded with the Sirt1 agonist SRT1720 accelerates the healing of diabetic ischemic ulcers.

Refractory ischemic ulcers that occur in patients with diabetes present a major clinical challenge. Embryonic artery cluster of differentiation 133+ cells (EACCs) may promote the healing of diabetic ulcers; however, the high glucose environment in the diabetic ulcers decreases the survival rate of transplanted EACCs and inhibit their biological function. Furthermore, microcirculation in diabetic ischemic ulcers is impaired, which inhibits the beneficial effect of EACCs. In the current study, the Sirt1 agonist SRT1720 was selected as a therapeutic drug and loaded into a dressing composed of PLGA, collagen and silk (PCSS) formed using electrospinning technology. EACCs were seeded onto the PCSS dressing and this was used to treat diabetic ulcers. The results indicated that SRT1720 promotes the proliferation of EACCs, enhances the secretion of vascular endothelial growth factor A, interluekin 8 and basic fibroblast growth factor, and inhibits the secretion of tumor necrosis factor α. Furthermore, SRT1720 promoted the paracrine function of EACCs and promoted the proliferation and migration of human umbilical vein endothelial cells. PCSS induced the steady release of SRT1720 over a 15-day period and PCSS seeded with EACCs (PCSS-EACCs) were transplanted into the diabetic ischemic ulcers of mice with diabetes. The results of these experiments indicated that angiogenesis and the healing of diabetic ischemic ulcers was significantly improved following the transplantation of PCSS-EACCs. Therefore, PCSS-EACCs may be a novel and effective treatment for diabetic ischemic ulcers.

An inflammatory memory and angiogenic self-assembling nanofiber hydrogel scaffold seeded with Akkermansia muciniphila to accelerate the healing of diabetic ischemic ulcers.

Refractory ulcers are a major challenge in the treatment of a diabetic foot, because of the immunodeficient, ischemic and high-glucose microenvironment. Inflammatory memory peptides, which were extracted from the immune mediator absent in melanoma 2 (AIM2), could effectively improve the immunodeficient microenvironment and special angiogenic peptides could effectively promote angiogenesis. Moreover, the gut flora Akkermansia muciniphila (A. muciniphila) participates in diabetic metabolism and could decrease high-glucose levels. In this research, a polypeptide skeleton (PPS) was synthesized based on 3,4-dihydroxyphenylalanine (DOPA) and peptides, forming the hydrophilic and hydrophobic parts. Inflammatory memory peptides and angiogenic peptides were synthesized and conjugated with the PPS, which then formed an anisotropic hydrogel through the self-assembling of ß-sheet peptides based on hydrophobicity and DOPA oxidation. A. muciniphila was seeded into the hydrogel and transported into diabetic ischemic ulcers through subcutaneous injection, and the healing of diabetic ischemic ulcers was promoted. The inflammatory memory peptides were released based on the A. muciniphila enzyme response, and they firstly improved the immunity of the local surroundings. Then, the angiogenic peptides were also released through irradiation and they promoted angiogenesis. Additionally, the transported A. muciniphila could decrease the local glucose levels and spontaneously regress once the diabetic ischemic ulcers had healed. A. muciniphila combined with a functional polypeptide hydrogel may be a novel strategy for diabetic ischemic ulcer treatment.

Effect of brain-derived neurotrophic factor on mesenchymal stem cell-seeded electrospinning biomaterial for treating ischemic diabetic ulcers via milieu-dependent differentiation mechanism.

Great challenges in transplantation of mesenchymal stem cells (MSCs) for treating ischemic diabetic ulcers (IDUs) are to find a suitable carrier and create a beneficial microenvironment. Brain-derived neurotrophic factor (BDNF), a member of neurotrophin family, is considered angiogenic and neuroprotective. Given that IDUs are caused by vascular disease and peripheral neuropathy, we used BDNF as a stimulant, and intended to explore the role of new biomaterials complex with MSCs in wound healing. BDNF promoted the proliferation and migration of MSCs using MTT, transwell, and cell scratch assays. The activity of human umbilical vein endothelial cells (HUVECs) was also enhanced by the MSC-conditioned medium in the presence of BDNF, via a vascular endothelial growth factor-independent pathway. Since proliferated HUVECs in the BDNF group made the microenvironment more conducive to endothelial differentiation of MSCs, by establishing co-culture systems with the two cell types, endothelial cells derived from MSCs increased significantly. A new biomaterial made of polylactic acid, silk and collagen was used as the carrier dressing. After transplantation of the BDNF-stimulated MSC/biomaterial complex, the ulcers in hindlimb ischemic mice healed prominently. More blood vessel formation was observed in the wound tissue, and more MSCs were co-stained with some endothelial-specific markers such as cluster of differentiation (CD)31 and von Willebrand Factor (vWF) in the treatment group than in the control group. These results demonstrated that BDNF could improve microenvironment in the new biomaterial, and induce MSCs to differentiate into endothelial cells indirectly, thus accelerating ischemic ulcer healing.

[The effect of heme oxygenase-1 on BMSCs damaged by high-concentration glucose].

This investigation was aimed to explore whether over-expression of 27heme oxygenase-1 (HO-1) could protect bone marrow mesenchymal stem cells(BMSCs)against injury induced by high-concentration glucose. We cultured BMSCs in high-concentration glucose medium, and up-regulated or inhibited HO-1 expression in BMSCs through its agonist or inhibitor. We detected the ability of BMSCs proliferation and secretion respectively by MTT and enzyme-linked immunosorbnent assay (ELISA). Then we detected the effect of BMSCs conditions medium on proliferation and migration of human umbilical vein endothelial cells (HUVECs) through scratch experiments and transwell assay. It was found that HO-1 over-expression could not only promote BMSCs proliferation, but also promote secretion of vascular endothelial growth factor (VEGF), and could further accelerate the proliferation and migration of HUVECs. It could be well concluded that HO-1-over-expressing BMSCs can not only inhibit damage induced by high-concentration glucose, but can promote the proliferation and migration of vascular endothelial cells through paracrine as well. The result indicated that HO-1-over-expressing BMSCs played an important role in the treatment of diabetic vascular complication.

The effect of heme oxygenase-1 complexed with collagen on MSC performance in the treatment of diabetic ischemic ulcer.

Diabetic ischemic ulcer is an intractable diabetic complication. Bone marrow mesenchymal stem cells (BMSCs) have great potential in variety of tissue repair. In fact, poor cell viability and tolerance limit their ability for tissue repair. In addition, it is difficult for stem cells to home and locate to the lesion. In this study, we explore whether over-expression of heme oxygenase-1 (HO-1) in BMSCs complexed with collagen play an important role in treatment of diabetic ischemic ulcers. In vitro, over-expression of HO-1 promoted the proliferation and paracrine activity of BMSCs and the conditioned medium of BMSCs accelerated HUVECs migration and proliferation. These processes were closely related to Akt signaling pathway and were not dependent on Erk signaling pathway. In vivo, in order to make BMSCs directly act on the wound, we choose a solid collagen as a carrier, BMSCs were planted into it, ischemic wounds of diabetic mice were covered with the complex of BMSCs and collagen. The results indicate that the complex of HO-1-overexpressing BMSCs and collagen biomaterials can significantly promote angiogenesis and wound healing. These preclinical findings open new perspectives for the treatment of diabetic foot ulcers.

Neurotrophin-3 accelerates wound healing in diabetic mice by promoting a paracrine response in mesenchymal stem cells.

Angiogenesis is a major obstacle for wound healing in patients with diabetic foot wounds. Mesenchymal stem cells (MSCs) have an important function in wound repair, and neurotrophin-3 (NT-3) can promote nerve regeneration and angiogenesis. We investigated the effect of NT-3 on accelerating wound healing in the diabetic foot by improving human bone marrow MSC (hMSC) activation. In vitro, NT-3 significantly promoted VEGF, NGF, and BDNF secretion in hMSCs. NT-3 improved activation of the hMSC conditioned medium, promoted human umbilical vein endothelial cell (HUVEC) proliferation and migration, and significantly improved the closure rate of HUVEC scratches. In addition, we produced nanofiber mesh biological tissue materials through the electrospinning technique using polylactic acid, mixed silk, and collagen. The hMSCs stimulated by NT-3 were implanted into the material. Compared with the control group, the NT-3-stimulated hMSCs in the biological tissue material significantly promoted angiogenesis in the feet of diabetic C57BL/6J mice and accelerated diabetic foot wound healing. These results suggest that NT-3 significantly promotes hMSC secretion of VEGF, NGF, and other vasoactive factors and that it accelerates wound healing by inducing angiogenesis through improved activation of vascular endothelial cells. The hMSCs stimulated by NT-3 can produce materials that accelerate wound healing in the diabetic foot and other ischemic ulcers.

The use of BDNF to enhance the patency rate of small-diameter tissue-engineered blood vessels through stem cell homing mechanisms.

The patency rate of small-diameter tissue-engineered blood vessels is the determinant for their application in coronary artery bypass grafting. The coronary artery is innervated by vagus nerves. The origin of vagus nerves is rich in brain-derived neurotrophic factors (BDNF). We have investigated whether BDNF could improve the patency rate of small-diameter tissue-engineered blood vessels through promoting stem cell homing and paracrine activity. In vitro, we isolated early and late endothelial progenitor cells (EPCs) and found BDNF could promote single clone formation and paracrine function of EPCs, and could also induce the proliferation, migration and differentiation of late EPCs. BDNF could enhance the capturing of EPCs in parallel-plate flow chamber. Flow cytometric analysis and laser-scanning confocal microscope showed BDNF could enhance the mobilization and homing of C57BL/6 mouse EPCs after wire injury. Based on it, BDNF was coupled to the lumen surface of the blood vessel matrix material incubated with collagen through SPDP to construct BDNF-modified small-diameter tissue-engineered blood vessel. The blood vessel patency rate was significantly higher than that of control group 8 weeks after implantation in rats and the endothelialization level was superior to control. These results demonstrate that BDNF can effectively improve patency of small-diameter tissue-engineered blood vessels through stem cell homing and paracrine, and it is expected to play an important role in the construction of substitutes for coronary artery bypass grafting.

Development of transgenic endothelial progenitor cell-seeded stents.

Endothelial progenitor cell (EPC)-seeded intravascular stents may reduce or prevent in-stent restenosis. A20 can play an important role for preventing vascular restenosis. Therefore, it is very important how to enhance the seeding efficiency of A20-modified EPCs on the stent for preventing in-stent restenosis. To approach this problem, we developed a novel transgenic EPC-seeded stent and evaluated its feasibility and efficiency. EPCs were isolated and purified from umbilical blood using immunomagnetic beads and then transfected with the A20 gene. One stent type (type 1) was coated with EDC cross-linked collagen, and another stent type (type 2) was coated with EDC cross-linked collagen and bound to the CD34 antibody using the bifunctional coupling agent N-succinmidyl3-(2-pyridyldithio) propionate (SPDP). Then, the stents were seeded with EPCs transfected with the A20 gene. The stents were implanted in biological artificial vessels, and cell adhesion was determined in a flow chamber. Cell growth was also measured. EPCs were transfected successfully with the A20 gene. The cells covered both types of stents with favorable biological function. After placement in a flow chamber, the number of cells attached to type 1 stents significantly dropped and their distribution was scattered. Type 2 stents were basically covered with cells and there were more cells on type 2 stents than on type 1 stents (p < 0.01). Collagen-coupled antibody effectively improves the seeding of transgenic EPCs, offering a new choice of stents to prevent restenosis caused by vascular disease after interventional treatment.

[Engineering and expression of sequence-specific DNA-binding zinc finger protein].

This experiment was aimed to create A20 gene site-specific zinc finger DNA-binding protein. The sequence of A20 gene promoter was analyzed with bioinformatics means and submitted to ZF Tools Server at TSRI. Using the database of the web site, we determined the A20 gene valid target sites and designed the amino acid sequence of zinc finger protein predicted to be bound to the target site. And then, the structure of the protein sequence was analyzed and homology was modeled with various bioinformatics means. Based on the characteristic of this protein, the prokaryotic expression vector pTYB11-ZFP was constructed and expressed. Thus, the artificial zinc finger protein that recognized A20 specific sequence was designed, and expressed in Escherichia coli. The results indicate that it is feasible to design engineered artificial Zinc finger proteins by means of bioinformatics.

Development of anti-atherosclerotic tissue-engineered blood vessel by A20-regulated endothelial progenitor cells seeding decellularized vascular matrix.

To investigate whether decellularized vascular tissues and A20-regulated endothelial progenitor cells can be used for constructing a transgenic tissue-engineered blood vessel with anti-atherosclerotic vascular stenotic properties. A20 gene-transfected endothelial progenitor cells differentiated endothelial cells and smooth muscle cells attached to and migrated into the decellularized porcine vascular scaffolding in a bioreactor. The histology of the conduits revealed viable and layered tissue. Scanning electron microscopy showed confluent, homogeneous tissue surfaces. The mechanical strength of the pulsed constructs was similar to that of the human artery. In vivo, the A20 gene-transfected tissue-engineered blood vessels were transplanted into the carotid artery of a rat for 6 months. Blood vessel xenotransplantation caused hyperacute rejection; all transplanted control blood vessels were completely rejected, but A20-transfected tissue-engineered blood vessels demonstrated good flow on implantation, and remained open for 6 months postoperatively, as assessed by Doppler. The HE stain demonstrated that the vessels were patent, without evidence of stenosis or dilatation after 6 months. These results demonstrate that transgenic tissue-engineered blood vessels have long-term patency and unique anti-stenotic properties.