Polyvinyl alcohol /chitosan membranes with copaiba oil emulsion and silver nanoparticles for possible application in wounds.

The interaction of copaiba oil in the polymer matrix of chitosan can produce a favorable synergistic effect and potentiate properties. Indeed, the bioactive principles present in copaiba oil have anti-inflammatory and healing action. In the present work, chitosan membranes containing different contents of copaiba oil copaíba (0.1, 0.5, 1.0 and 5.0% (v/v)) were for the first time investigated. The membranes were developed by the casting method and analyzed for their morphology, degree of intumescence, moisture content, contact angle, Scanning Electron Microscope, and X-ray diffractometry. These chitosan/copaiba oil porous membranes disclosed fluid absorption capacity, hydrophilic surface, and moisture. In addition, the results showed that chitosan membranes with the addition of 1.0% (v/v) of copaiba oil presented oil drops with larger diameters, around 123.78 μm. The highest fluid absorption indexes were observed in chitosan membranes containing 0.1 and 0.5% (v/v) of copaiba oil. In addition, the copaiba oil modified the crystalline structure of chitosan. Such characteristics are expected to favor wound treatment. However, biological studies are necessary for the safe use of chitosan/copaiba oil membrane as a biomaterial.

The calcium-dependent regulation of spheroid formation and cardiomyogenic differentiation for MSCs on chitosan membranes

Multicellular three-dimensional (3D) spheroids allow intimate cell-cell communication and cell-extracellular matrix interaction. Thus, 3D cell spheroids better mimic microenvironment in vivo than two-dimensional (2D) monolayer cultures. The purpose of this study was to evaluate the behaviors of human dental pulp cells (DPCs) cultured on chitosan and polyvinyl alcohol (PVA) membranes. The protein expression of hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor (VEGF), and the migration ability of the DPCs from 2D versus 3D environments were investigated. The results showed that both chitosan and PVA membranes support DPCs aggregation to form multicellular spheroids. In comparison to 2D cultures on tissue culture polystyrene, DPC spheroids exhibited higher protein expression of HIF-1α and VEGF. The treatment with YC-1 (inhibitor to HIF-1α) blocked the upregulation of VEGF, indicating a downstream event to HIF-1α expression. When DPC spheroids were collected and subjected to the transwell assay, the cells growing outward from 3D spheroids showed greater migration ability than those from 2D cultures. Moreover, DPCs aggregation and spheroid formation on chitosan membrane were abolished by Y-27632 (inhibitor to Rho-associated kinases), whereas the inhibitory effect did not exist on PVA membrane. This suggests that the mechanism regulating DPCs aggregation and spheroid formation on chitosan membrane is involved with the Rho-associated kinase signaling pathway. In summary, the multicellular spheroid structure was beneficial to the protein expression of HIF-1α and VEGF in DPCs and enhanced the migration ability of the cells climbing from spheroids. This study showed a new perspective in exploring novel strategies for DPC-based research and application.

Electrospinning was used as an effective route to pattern chitosan (CS) and polycaprolactone (PCL) membranes with submicron fibers having different chemical structure (PCL or PCL/collagen) and physical characteristics (size: between ≈200 and 550 nm; randomly oriented or aligned form). While the PCL fibers with diameters in the same range (≈200 nm) were patterned on both of CS and PCL membranes to evaluate the influence of the underlying membrane chemistry, only CS membranes were patterned with PCL fibers having different sizes simply by changing the electrospinning conditions to investigate the effects of pattern characteristics. Furthermore, collagen was added to the PCL fiber structure to change the chemical composition of the fibers in a cell-attractive way. Two cell lines with different morphologies, fibroblastic MC3T3-E1 preosteoblasts and epithelial Madine Darby Bovine Kidney (MDBK) cells, were cultured on the patterned membranes. The observation of cellular behavior in terms of cell morphology and F-actin synthesis was realized by scanning electron microscopy and confocal microscopy analysis during the first 12 h of culture period. The viability of cells was controlled by MTT assay through 96 h of cell culture. The cell culture studies indicated that the leading aspect for the morphology change on patterned membranes was the fiber orientation. The aligned topography controlled the morphology of cells both on CS and PCL membranes. In the presence of collagen in the fiber structure, F-actin filament synthesis increased for MC3T3-E1 and MDBK cell lines.

A comparative study on the chitosan membranes prepared from glycine hydrochloride and acetic acid

To prepare nano polypyrrole (PPy)/chitin composite membrane and observe their biocompatibility. The nano PPy was synthesized by microemulsion polymerization, blended with chitosan and then formed membranes. The membranes were then modified by acetylation to get the experimental membranes (nano PPy/chitin composite membranes, group A). The chitosan membranes (group B) and chitin ones (group C) modified by acetylation acted as control. Scanning electron microscopy and FT-IR spectra were used to identify the nano PPy and the membranes of each group. And the conductivity of membranes of each group was measured. Schwann cells were co-cultured in vitro with each group membranes to observe the biocompatibility by inverted microscope observing, living cell staining, cell counting, and immunofluorescence staining. The lysozyme solution was used to evaluate the degradation of the membranes in vitro. The FT-IR spectra showed that the characteristic vibrational absorption peaks of C=C from nano PPy appeared at 1 543.4 cm -1 and 1 458.4 cm -1. Scanning electron microscopy observation revealed that the size of nano PPy particles was about 100-200 nm. The nano PPy particles were synthesized. It was successful to turn chitosan to chitin by the acetylation, which was investigated by FT-IR analysis of membranes in groups A and C. The characteristic peaks of the amide Ⅱ band around 1 562 cm -1 appeared after acetylated modification. Conductivity test showed that the conductivity of membranes in group A was about (1.259 2±0.005 7)×10 -3 S/cm, while the conductivity of the membranes in groups B and C was not detected. The nano PPy particles uniformly distributed on the surface of membranes in group A were observed by scanning electron microscope; the membranes in control groups were smooth. As a result, the nano PPy/chitin composite membranes with electrical conductivity were obtained. The cultured Schwann cells were found to survive with good function by fluorescein diacetate live cell staining, soluble protein-100 immunofluorescence staining, and inverted microscope observing. The cell counting showed that the proliferation of Schwann cells after 2 days and 4 days of group A was more than that of the two control groups, and the differences were significant ( P<0.05). It indicated that the nano PPy/chitin composite membranes had better ability of adhesion and proliferation than those of chitosan and chitin membranes. The degradation of membranes in vitro showed that the degradation rates of membranes in groups A and C were significantly higher than those in group B at all time points ( P<0.05). In a word, the degradation performance of the membranes modified by acetylation was better than that of chitosan membranes under the same condition. The nano PPy and chitosan can be blended and modified by acetylation successfully. Nano PPy/chitin composite membranes had electrical conductivity, degradability, and good biocompatibility in vitro.

Synthesis and characterization of modified chitosan membranes for applications in electrochemical capacitor

Biologically active sodium pentaborate pentahydrate and Hypericum perforatum oil loaded polyvinyl alcohol: chitosan membranes

Objective To study the feasibility of using chitosan membrane to carry and transport melanocytes, in order to refine the technique for melanocyte transplantation with chitosan membrane. Methods Melanocytes were inoculated onto chitosan membrane and cultured for a period of time, then, electron microscopy,MTT assay and NaOH assay were carried out to estimate the adherence, growth and melanogenesis of the melanocytes. Skin wound surface was prepared in 12 nude mice, which were equally divided into 3 groups, test group inoculated with melanocytes on chitosan membrane, negative control group I treated with chitosan membrane without melanocytes, and negative control group II directly dressed immediately after the preparation of wound surface. On day 10 and 20 after the transplantation, confocal laser microscopy and immunohistochemistry were performed to observe the migration of melanocytes into the skin wound surface. Results Scanning electron microscopy and inverted microscopy showed that melanocytes were evenly distributed on and adhered well to the underlying chitosan membrane. As the growth curve of melanocytes demonstrated, chitosan membrane could support the normal growth of melanocytes, and no significant difference was observed in the synthesized melanin content between melanocytes cultured on the chitosan membrane and those in culture disks (0.087 ± 0.027 vs. 0.101 ± 0.036, t = 0.79, P > 0.05). Melanocytes were seen at the transplantation sites by confocal laser microscopy, and biopsy specimens from the transplantation sites stained positive for antimelan-A monoclonal antibody. Conclusions Melanocytes can adhere to and grow on the chitosan membrane,which can facilitate the migration of melanocytes to the transplantation sites in animals with the maintenance of biological activity of melanocytes. Key words: Chitosan; Membranes; Melanocytes; Transplantation

Papain (an enzyme from the latex of Carica papaya) is an interesting natural bioactive macromolecule used as therapeutic alternative for wound healing due to debridement action in devitalized or necrotic tissues. However, its use in high doses can induce potential skin irritation and side effects. In this study, experiments explored the ability of chitosan membrane to immobilize papain, consequently improving enzymatic activity and controlling enzyme release. Papain-loading capacity was tested via experiments of force microscopy (AFM), scanning electron microscopy (SEM-FEG), and X-ray diffraction analyses. Fourier transform infrared spectroscopy and thermal analyses assessed the enzyme interactions with the copolymer. The investigation of the feasibility of membranes included pH on the surface, elasticity, and breaking strength measurements. The surface wettability and swelling capacity of different formulations revealed the best formulation for in vitro papain release experiments. The membranes had a transparent, rough, crystalline characteristic, which was homogeneous with the membrane within the neutrality. The immobilization of papain in the chitosan membrane resulted in a decrease in the vibration band characteristic of pure papain, suggesting a displacement in the vibration bands in the FTIR spectrum. The presence of papain decreased hydrophobicity on the surface of the membrane and disturbed the membrane's ability to swell. Chitosan membranes containing papain 2.5% (0.04 g) and 5.0% (0.08 g) preserved feasible properties and improved the enzymatic activity compared (0.87 ± 0.12 AU/mg and 1.59 ± 0.10 AU/mg) with a free papain sample (0.0042 ± 0.001 AU/mg). Concentrations of over 10% (0.16 g) led to phase separation into membranes. Chitosan membranes exhibited a slow papain release behavior adjusted via the Higushi model. The experimental achievements suggest a novel and promising method for the enhancement of papain. The results indicate the potential for prolonged bioactivity for use on wounds.

Cu-MOF loaded chitosan based freeze-dried highly porous dressings with anti-biofilm and pro-angiogenic activities accelerated Pseudomonas aeruginosa infected wounds healing in rats

Objectives: The objective of this study is to compare the bone-regenerating capacity between chitosan foam and chitosan membrane scaffolds. Methods: A medium-weight chitosan acidic mixture was used to prepare two scaffolds of freeze-dried chitosan foam (CF). One of the two CF scaffolds was physically crosslinked by NaHCO3 to obtain chitosan membrane (CM). A morphological assessment of the specimens' porosity was carried out by scanning electron microscopy (SEM). An MTT assay of the CM and CF specimens using rats' bone marrow mesenchymal stem cells (MSCs) was carried out. Then, 38 albino rats were subjected to surgical implantation in a critical-size defect of the femur bone. The rats were divided into three groups according to the type of implanted scaffold (Control (no scaffold) n = 10, CM (chitosan membrane) n = 14, CF (chitosan foam) n = 14). Each group was equally subdivided into two subgroups according to the time of euthanasia (21 d, 35 d). The femur bones were dissected for a histological analysis (hematoxylin and eosin, and Masson trichrome). The results of the histological analysis were graded according to a scoring system. A statistical analysis of the pore size and histological grading was carried out. Results: CF had a higher mean pore size (65.42 µm) compared to CM (6.44 µm); CM showed a significantly higher proliferation of MSCs at 72 h. Both the CM and CF groups showed a significantly higher bone regeneration and lower inflammation than the control group. The CF group showed a significantly higher bone regeneration score than the CM group, especially at 35 d with more dense compact lamellar bone structure. Conclusions: The higher mean pore size of CF allowed for a higher bone regenerating capacity than the crosslinked CM.

Replica Microscopy and Scanning Electron Microscopy of Laser Impacts on the Skin

Modification of chitosan membrane with poly(vinyl alcohol) and biocompatibility evaluation

Characterization of the modified chitosan membrane cross-linked with genipin for the cultured corneal epithelial cells