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Treating burn-related injuries remains challenging, because of donor site morbidity, bacterial infection, and major scarring. Recent advances have led to the development of promising natural and synthetic polymer scaffolds, the objective of which is to regenerate skin using a “top-down” approach, in which the top layers of the skin populated by fibroblasts and keratinocytes are regenerated first. An often-overlooked component of the integumentary system is the subcutaneous adipose tissue, which has been implicated in a variety of activities, including wound healing. We have created a novel coating composed of collagen VI (col VI) and heparin sulfate (HS) that promotes adipocyte attachment and adipogenesis at a rate significantly faster at both early and late time points (5 and 23 days), compared to a noncoated control. In addition, Vicryl, which is a biodegradable surgical implant mesh, was coated with the optimized col VI–HS ratio. Coated Vicryl had an increased number of attached adipocytes with more-pr...

The modification of proteins with reactive handles has facilitated the use of these biomolecules in diverse fields including drug delivery, diagnostics, environmental remediation, and cell culture matrices. Recently, a new “click” type reaction, sulfur(VI) fluoride exchange (SuFEx), was reported between sulfuryl fluoride and various organic moieties (e.g. hydroxyl, amine) to yield a sulfamoyl fluoride ( NSO 2 F) or fluorosulfate ( OSO 2 F) moiety under biphasic conditions. The model protein bovine serum albumin (BSA) was reacted with SO 2 F 2 gas under biphasic conditions to yield SO 2 F functionalized protein. The resultant BSA-SO 2 F was characterized using gel electrophoresis, mass spectroscopy and Fourier transmission infrared spectroscopy to confirm the addition of SO 2 F functional group. SuFEx modification of BSA caused a marked change in the pH dependent size and zeta potential of the protein as well as increased the protein’s denaturation temperature. Crucially, BSA-SO 2 F was demonstrated to be biocompatible after 72 h incubation with A549 lung endothelial cells. Due to the unique and elective reactivity of the SO 2 F group with amines, BSA-SO 2 F could be self-condensed to form a biocompatible hydrogel that was used in co-culture with HEK 293 cells. In addition, polyethylene glycol (PEG) with reactive OSO 2 F groups at chain end was conjugated with BSA under various pH conditions through SuFEx chemistry. This communication, to our knowledge, is the first report of the application of the SuFEx in the field of bioconjugates.

Post-polymerization modification of polymer chain-ends is a powerful tool for the preparation of functional materials. “Click” type reactions represent the most prevalent post-polymerization strategies. We applied the recently described sulfur(VI) fluoride exchange reaction to functionalize the chain-ends of four polymers prepared using activators generated by electron transfer atom transfer radical polymerization. Aryl-TBDMS terminated polymers were reacted with aryl sulfurofluoridate compounds with a near quantitative conversion. This research represents the application of a new click type reaction to prepare functional polymers.

Biopolymer–ceramic composites are thought to be particularly promising materials for bone tissue engineering as they more closely mimic natural bone. Here, we demonstrate the fabrication by electrospinning of fibrous chitosan-hydroxyapatite composite scaffolds with low (1 wt %) and high (10 wt %) mineral contents. Scanning electron microscopy (FESEM), energy dispersive spectroscopy (EDS) and unidirectional tensile testing were performed to determine fiber surface morphology, elemental composition, and tensile Young's modulus (E) and ultimate tensile strength (σUTS), respectively. EDS scans of the scaffolds indicated that the fibers, crosslinked with either hexamethylene-1,6-diaminocarboxysulfonate (HDACS) or genipin, have a crystalline hydroxyapatite mineral content at 10 wt % additive. Moreover, FESEM micrographs showed that all electrospun fibers have diameters (122–249 nm), which fall within the range of those of fibrous collagen found in the extracellular matrix of bone. Young's modulus and ultimate tensile strength of the various crosslinked composite compositions were in the range of 116–329 MPa and 2–15 MPa, respectively. Osteocytes seeded onto the mineralized fibers were able to demonstrate good biocompatibility enhancing the potential use for this material in future bone tissue engineering applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3201–3211, 2015.

Chitosan is a naturally occurring polysaccharide, which has proven to be an attractive candidate for bone tissue engineering, due to its ability to promote osteoblast mineralization. Electrospinning has become a well-established cell scaffold processing technique, as it produces a high surface area to volume fibrous material that can mimic the three dimensionality of the extracellular matrix of a cell. In this study, we have investigated the osteoblast response to two different chemically crosslinked (hexamethylene-1,6-diaminocarboxysulfonate (HDACS) and genipin) electrospun chitosan scaffolds and their film counterparts in order to determine how material chemistry influences cellular behavior in conjunction with material topology. In addition, material properties of each fiber scaffold such as porosity and tensile strength were considered. MLO-A5 osteoblast cells grown on chitosan-HDACS scaffolds were found to display a more organized cellular network, along with significantly more filopodia extensions, compared to those grown on chitosan-genipin scaffolds. After 2 days of growth on chitosan-HDACS fibers, a higher level of alkaline phosphatase expression in MLO-A5 cells was reported compared to that of either chitosan-genipin fibers or films. These results indicate that not only chemistry, but also surface topology is an important effecter of cellular behavior. Ultimately, chitosan-HDACS fiber scaffolds provided an adequate substrate for osteoblast attachment and proliferation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3026–3033, 2015.

The rate of polarised secretion of sialosyl Lewis(a)(19-9) molecular species (SiaLeams) by SW1116 colorectal carcinoma cells is stimulated at least ninefold by the presence of 3 microM retinoic acid (RA). In order to investigate the intracellular origins of this augmentation, carcinoma cell membranes, membrane subfractions, and media were studied to determine alterations in sialosyl Lewis(a) levels, oligosaccharide composition, and core structures accompanying the capacity to increase export of this epitope. We observed a nine- to twentyfold increase in sialosyl Lewis(a) epitope levels in a light membrane subfraction from RA-treated cells. Antigenic molecules of < 200,000 M(r) on acrylamide gradient gels were concentrated in two doublets in the apparent M(r) range 106,000-152,000 on Western blots. Carbohydrate analyses of oligosaccharides from SiaLeams of membrane subfractions and apical media indicated much higher fucose/mannose, fucose/sialic, fucose/sialosyl Lewis(a), fucose/total CHO, and (3H) fucose incorporation in control samples than RA samples. Western blots of samples from membrane subfractions and media indicated that, in contrast to the effect of RA on the sialosyl Lewis(a) epitope, RA treatment did not augment cysteine-rich, PDTRP, blood group H-2, blood group A, and EGF receptor-like region epitopes in the media. In addition, Northern blots using the Lewis fucosyl transferase (FTIII) cDNA showed a dramatic diminution of mRNA encoding FTIII but apparently unaltered levels of sialyl transferase (ST4) mRNA. Since subterminal fucosylation of lactosyl termini blocks terminal sialylation, we conclude that one mechanism of sialosyl Lewis(a) induction in this culture system is the lower expression of the Lewis fucosyl transferase mRNA. Therefore less subterminal fucosylation of GlcNAc permits the prior sialylation of terminal Gal beta 1-3 moieties at oligosaccharide termini destined for export from the Golgi.

The lungs and hearts of young rats exposed to 100% oxygen (O2) for 8 days (27 to 35 days of age) were studied following recovery in room air at 60 days of age using morphometric, biochemical, and physiological techniques. In an attempt to prevent chronic oxygen toxicity 153 rats had transtracheal catheters surgically implanted and were treated during the O2 exposure with daily intratracheal injections of liposome-encapsulated superoxide dismutase (SOD) and/or catalase (CAT). Oxygen exposure in this model results in chronic cardiopulmonary alterations which include pulmonary hypertension, right ventricular hypertrophy, and a decrease in number of pulmonary arterioles 25 to 50 microns in diameter with increased muscularization of their walls. The volume densities of the parenchyma, parenchymal air space, and the alveolar space are increased, while that of the combined alveolar ductal and respiratory bronchiolar space is decreased. Daily intratracheal administration of liposome-encapsulated CAT (160 U) during the O2 exposure prevented these chronic changes. Liposome-encapsulated SOD (110 U) or SOD (50 U) + CAT (70 U) did not appear to have a preventive effect. During the first 3 to 5 days following oxygen exposure the lung tissue enzymes SOD, CAT, and glutathione peroxidase markedly increased. We conclude that in the young rat animal model liposome-encapsulated CAT (160 U) given intratracheally during the period of O2 exposure is safe and will prevent the chronic vascular and parenchymal damage due to oxygen toxicity.

We have used cDNAs coding for novel ADP-ribosylation factor-like molecules (ARL184 and ARL184Δ) to alter 19-9 antigen glycoprotein secretion in cultured human colorectal carcinoma cells SW1116 by transfection and cloning. This ARL contains a lipophilic N-terminal with an isoleucyl and 3 leucyl residues, 4 functioning consensus sequence GTP binding sites, and 184 total aminoacyl residues. An ARL cDNA was also constructed deleting the codon for the N-terminal glycyl moiety. The resulting cell clones were shown by Northern blots to overexpress ARL mRNA. Electron microscopy–immunocytochemistry also indicated the overexpression of ARL granules subcellularly. Secretion of the tumor-associated 19-9 antigen into apical medium was decreased 3- to 5-fold and the secretion of TCA/PTA precipitable 3 H-labeled glycoprotein was decreased by 34% in clone SW1116(ARL184)Δ. Western blot analyses of cell homogenates and media were in agreement with the secretion assays and showed a diminution of 170–200 kDa, 19-9, antigenicity in transfected cells and their media. Apical secretion of 19-9 antigen was diminished 14-fold in cells, SW1116 (ARL184)α, transfected with the complete ARL cDNA sequence, suggesting that the glycyl moiety may be required for maximal abatement. However, incorporation of label from [ 3 H]myristate into 22-kDa bands of NP-40 extracts and ARL-antigenic molecules of parent cells was 3-fold greater than that in samples from the two transfectants; thus the transfected cells may not myristylate the overexpressed ARL efficiently. Notwithstanding the N-terminal glycyl moiety undergoing some other modification, we conclude that overexpression of this ARL is sufficient to generate a 19-9-deficient phenotype. These ARLs may eventually disrupt terminal oligosaccharide glycosylation, resulting in an apparent diminished exocytosis of 19-9 glycoprotein carriers by transfected and cloned cells.