Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.     

Simple Patterned Nanofiber Scaffolds and Its Enhanced Performance in Immunoassay

Cancer has become the leading cause of death worldwide; early diagnosis and treatment of cancers is critical for the survival of the patients. The concentration of cancer markers in easy-to-access biological fluids can provide great assistance in screening for occult primary cancers, distinguishing malignant from benign findings, determining prognosis and prediction for cancer patients. The multiplex detection technology of a panel of cancer markers can greatly increase the accuracy of disease diagnosis. Herein, we briefly fabricate a high-throughput micro-immunoassay based on the electrospun polystyrene (PS) substrates to improve detection sensitivity. The immunoassay was evaluated by analyzing three different cancer biomarkers (AFP, CEA, VEGF). For AFP, CEA, VEGF immunofluorescence assay, the LOD of assay conducted on electrospun PS substrates before or after plasma and the conventional PS substrates were 0.42, 0.10, 1.12 ng/mL, 0.57, 0.09, 1.24 ng/mL, and 159.75, 26.19, 385.59 pg/mL, respectively (P < 0.05). Due to the high porosity and large surface area-to-volume ratio which is the foremost merit of nanostructures, and the plasma treatment which make the hydrophobic PS nanofibers hydropholic, the nanofibers substrates showed sufficient retention of immunoassay functionality and high potential for capture molecules immobilization. Consequently, the immunofluorescence assay conducted on electrospun PS substrates could significantly enhance the sensitivity and limits of detection.     

Silver-Zinc Redox-Coupled Electroceutical Wound Dressing Disrupts Bacterial Biofilm

Pseudomonas aeruginosa biofilm is commonly associated with chronic wound infection. A FDA approved wireless electroceutical dressing (WED), which in the presence of conductive wound exudate gets activated to generate electric field (0.3–0.9V), was investigated for its anti-biofilm properties. Growth of pathogenic P. aeruginosa strain PAO1 in LB media was markedly arrested in the presence of the WED. Scanning electron microscopy demonstrated that WED markedly disrupted biofilm integrity in a setting where silver dressing was ineffective. Biofilm thickness and number of live bacterial cells were decreased in the presence of WED. Quorum sensing genes lasR and rhlR and activity of electric field sensitive enzyme, glycerol-3-phosphate dehydrogenase was also repressed by WED. This work provides first electron paramagnetic resonance spectroscopy evidence demonstrating that WED serves as a spontaneous source of reactive oxygen species. Redox-sensitive multidrug efflux systems mexAB and mexEF were repressed by WED. Taken together, these observations provide first evidence supporting the anti-biofilm properties of WED.     

Potential of Magnetic Nanofiber Scaffolds with Mechanical and Biological Properties Applicable for Bone Regeneration

Magnetic nanofibrous scaffolds of poly(caprolactone) (PCL) incorporating magnetic nanoparticles (MNP) were produced, and their effects on physico-chemical, mechanical and biological properties were extensively addressed to find efficacy for bone regeneration purpose. MNPs 12 nm in diameter were citrated and evenly distributed in PCL solutions up to 20% and then were electrospun into nonwoven nanofibrous webs. Incorporation of MNPs greatly improved the hydrophilicity of the nanofibers. Tensile mechanical properties of the nanofibers (tensile strength, yield strength, elastic modulus and elongation) were significantly enhanced with the addition of MNPs up to 15%. In particular, the tensile strength increase was as high as ∼25 MPa at 15% MNPs vs. ∼10 MPa in pure PCL. PCL-MNP nanofibers exhibited magnetic behaviors, with a high saturation point and hysteresis loop area, which increased gradually with MNP content. The incorporation of MNPs substantially increased the degradation of the nanofibers, with a weight loss of ∼20% in pure PCL, ∼45% in 10% MNPs and ∼60% in 20% MNPs. Apatite forming ability of the nanofibers tested in vitro in simulated body fluid confirmed the substantial improvement gained by the addition of MNPs. Osteoblastic cells favored the MNPs-incorporated nanofibers with significantly improved initial cell adhesion and subsequent penetration through the nanofibers, compared to pure PCL. Alkaline phosphatase activity and expression of genes associated with bone (collagen I, osteopontin and bone sialoprotein) were significantly up-regulated in cells cultured on PCL-MNP nanofibers than those on pure PCL. PCL-MNP nanofibers subcutaneously implanted in rats exhibited minimal adverse tissue reactions, while inducing substantial neoblood vessel formation, which however, greatly limited in pure PCL. In vivo study in radial segmental defects also signified the bone regeneration ability of the PCL-MNP nanofibrous scaffolds. The magnetic, bone-bioactive, mechanical, cellular and tissue attributes of MNP-incorporated PCL nanofibers make them promising candidate scaffolds for bone regeneration.     

Cell Population Kinetics of Collagen Scaffolds in Ex Vivo Oral Wound Repair

Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.     

Self-Assembling Peptide Nanofiber Scaffolds Enhance Dopaminergic Differentiation of Mouse Pluripotent Stem Cells in 3-Dimensional Culture

Dopaminergic differentiation of embryonic stem cells (ESCs) gains more and more attention worldwide owing to its potential use for neurorestorative therapy for the treatment of Parkinson’s disease. The conventional 2D cell culture on petri dishes with various animal derived substrata such as collagen gels, laminin, and Matrigel is widely used to induce dopaminergic differentiation and it may limit the efficiency in the generation of dopaminergic neurons from ESCs and prevent their application for human therapies. Here, we reported that a self-assembling peptide made from natural amino acids has a property to generate a true 3D environment for dopaminergic differentiation. Mouse ESCs (R1) and mouse iPSCs (TTF-1) embedded in RADA16-I peptide-derived nanofiber scaffolds led to a marked increase in dopaminergic differentiation compared to the laminin-coated 2D culture or Matrigel-encapsulated 3D culture. These differentiated neurons expressed specific dopaminergic markers and produced appropriate patterns of action potential firing. Consistent with the increase in the number of dopaminergic neurons differentiated from R1 or TTF-1 in the self-assembling peptide nanofiber scaffold (SAPNS), both the expression levels of genes that involve in dopaminergic differentiation and maturation and the dopamine release in SAPNS culture were significantly elevated. The results of the study suggest that SAPNS provides a promising 3D culture system for dopaminergic differentiation.     

GNPs-CS/KGM as Hemostatic First Aid Wound Dressing with Antibiotic Effect: In Vitro and In Vivo Study

Ideal wound dressing materials should create a good healing environment, with immediate hemostatic effects and antimicrobial activity. In this study, chitosan/konjac glucomannan (CS/KGM) films embedded with gentamicin-loaded poly(dex-GMA/AAc) nanoparticles (giving GNP-CS/KGM films) were prepared as novel wound dressings. The results revealed that the modified CS/KGM films could be used as effective wound dressings and had significant hemostatic effects. With their microporous structure, the films could effectively absorb water from blood and trap blood cells. The gentamicinloaded poly(dex-GMA/AAc) nanoparticles (GNPs) also further promoted blood clotting, with their favorable water uptake capacity. Thus, the GNP-CS/KGM films had wound healing and synergistic effects that helped to stop bleeding from injuries, and also showed good antibiotic abilities by addition of gentamicin to the NPs. These GNPCS/KGM films can be considered as promising novel biodegradable and biocompatible wound dressings with hemostatic capabilities and antibiotic effects for treatment of external bleeding injuries.     

Evaluation of Lyophilised, Gamma-Irradiated Amnion as a Biological Dressing

Burns, non-healing wounds and pressure sores cause extensive damage to the skin leading to infection and loss of precious body fluids. Despite advances in burn management the mortality rate continues to be high and the search for an economical and easily available dressing to control burn wound infection continues. Autologous skin has limited availability and is associated with additional scarring. Conventional dressings require frequent changes which can be painful and may even require anaesthesia.Amnion is an excellent biological dressing and its use in the treatment of burns has special appeal in India as there are religious barriers to the acceptance of bovine and porcine skin.Lyophilised, irradiated amnion provided for the first time in the country by the Tata Memorial Hospital Tissue Bank was evaluated as a temporary biological dressing. It was used to treat 35 patients with burns, 21 patients with bedsores and non-healing ulcers and the skin graft donor sites of 11 patients.The amnion was easy to handle and stuck well to the raw wound bed. An open dressing was used in most of the second degree burns which healed with hyperemia and early pigmentation. In patients with third degree burns, ulcers or skin graft donor sites, closed dressings were used. The exudate and induration were reduced and patients were more comfortable and experienced less pain. There was healthy granulation with good re-epithelialisation. Amnion was not used in patients with infected third degree burns.     

Protein Synthesis as a Requirement for Wound Xylem Differentiation

An increasing rate of protein synthesis was observed during the first 2 days after the isolation of 2 mm thick internodal stem slices of Coleus on a sucrose-agar medium. This rise in the rate of protein synthesis preceeded the first visible signs of wound-vessel member differentiation in the cultured stem slices. Irradiation of tissue slices with 4000 R of x-rays at isolation reduced the numbers of wound-vessel members differentiated after 7 days in culture by 51 per cent, and this level of x-irradiation was observed to inhibit protein synthesis by the cultured stem slices. Treatment of the tissue slices with exogenous auxin (0.05 mg/1) after irradiation did not alter the degree of inhibition of xylem differentiation. Actinomycin D inhibited wound-vessel member differentiation, but it had no effect on the endogenous growth of the cultured stem slices. Similarly, auxin at 0.05 mg/1 was without effect on the endogenous growth rate of the stem slices. Actinomycin D treatment was highly effective in inhibiting xylem differentiation if it was supplied to the tissues within the first 48 hours after isolation; actinomycin D treatment had no significant effect on xylem differentiation when it was given after the first 2 days of culture. Chloramphenicol (10^?3M) inhibited both xylem differentiation and the endogenous growth of the cultured stem slices.     

Material self-assembly as a physicochemical process

Material self-assembly as exemplified in protobiogeneses is shown to be a result of molecular exchange interaction in reacting chemicals. Temporally and spatially correlated aggregates of reacting chemicals work as vertices of molecular exchange interaction. If the material accumulation rate at a locally correlated aggregate spontaneously happens to become positive at a certain time, the material accumulation due to self-assembly will increase with time afterward. A spontaneous formation of looped reaction at locally correlated aggregates of reacting chemicals can initiate a material self-assembly at succeeding times.     

创面生物活性玻璃修复材料促进家猪创面愈合的实验研究

目的：观察创面生物活性玻璃修复材料对家猪皮肤创面的促愈合作用。方法：选择14头家猪,随机分成7组,每组2头,在每头猪的脊柱两旁制造3个4×4cm的全层皮肤缺损的创面模型,每头猪6个创面又分成实验组和空白对照组,于试验后每天观察创面愈合情况,第1、3、7、14、21、28、35天图像分析计算创面愈合率,并同时取创面组织行组织学染色,观察各组材料对家猪皮肤全层缺损创面愈合的影响。结果：在涂材料的实验组和空白对照组创面愈合时间分别是23.19±1.27d、29.52±1.54d两组组间比较,具有统计学意义（P〈0.05）;实验组的创面愈合率在各时间段均高于空白对照组,差异有统计学意义（P〈0.05）;组织学观察实验组的上皮化程度、表皮生长、成纤维细胞、毛细血管数量均好于空白对照组。结论：创面生物活性玻璃修复材料对家猪皮肤创面愈合具有促进作用,可作为一种新型的促愈合覆盖材料进一步研究。     

Gentamicin-Loaded Wound Dressing With Polyvinyl Alcohol/Dextran Hydrogel: Gel Characterization and In Vivo Healing Evaluation

To develop a gentamicin-loaded wound dressing, cross-linked hydrogel films were prepared with polyvinyl alcohol (PVA) and dextran using the freezing–thawing method. Their gel properties such as gel fraction, swelling, water vapor transmission test, morphology, tensile strength, and thermal property were investigated. In vitro protein adsorption test, in vivo wound healing test, and histopathology were performed. Dextran decreased the gel fraction, maximum strength, and thermal stability of hydrogels. However, it increased the swelling ability, water vapor transmission rate, elasticity, porosity, and protein adsorption. The drug gave a little positive effect on the gel properties of hydrogels. The gentamicin-loaded wound dressing composed of 2.5% PVA, 1.13% dextran, and 0.1% drug was more swellable, flexible, and elastic than that with only PVA because of its cross-linking interaction with PVA. In particular, it could provide an adequate level of moisture and build up the exudates on the wound area. From the in vivo wound healing and histological results, this gentamicin-loaded wound dressing enhanced the healing effect more compared to conventional product because of the potential healing effect of gentamicin. Thus, this gentamicin-loaded wound dressing would be used as a potential wound dressing with excellent forming and improved healing effect in wound care.     

CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification

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Jasmonates as Signals in the Wound Response

Plant responses to wounding and herbivore attack are orchestrated by complex signaling pathways that link the production of chemical and physical signals at the wound site to activation of gene expression and other cellular processes. The systemic nature of many wound-induced responses provides an attractive opportunity to study intercellular signaling pathways that operate over long distances within the plant. Genetic dissection of the wound-response pathway in tomato indicates that (1) systemin and its precursor protein, prosystemin, are upstream components of an intercellular signaling cascade that requires the biosynthesis and action of jasmonic acid (JA); and (2) physiological processes regulated by this pathway confer host resistance to a broad spectrum of plant invaders. Grafting experiments conducted with mutants defective in systemic wound signaling indicate that systemin functions at or near the wound site to trigger the production of JA, which in turn acts non-cell autonomously to promote systemic defense responses. The location of JA biosynthetic enzymes within the companion cell-sieve element complex of vascular bundles, together with the accumulation of JA in vascular tissues, support a role for jasmonates as phloem-mobile signals. The recent discovery of enzymes involved in the metabolism of JA to volatile methyl-JA and bioactive JA-amino acid conjugates has potential implications for the mechanism by which JA promotes wound signaling. Species-specific differences in the mechanism of wound signaling appear to reflect the way in which the wound-induced jasmonate pathway is regulated by other signals including systemin, cell wall-derived oligosaccharides, ethylene, and insect-derived elicitors. Adding to the complexity of the wound-induced jasmonate cascade are wound-signaling pathways that operate independently of JA.