Abstract An injectable tissue-engineered adipose substitute that could be used to deliver adipose-derived stem cells (ASCs), filling irregular defects and stimulating natural soft tissue ...regeneration, would have significant value in plastic and reconstructive surgery. With this focus, the primary aim of the current study was to characterize the response of human ASCs encapsulated within three-dimensional bioscaffolds incorporating decellularized adipose tissue (DAT) as a bioactive matrix within photo-cross-linkable methacrylated glycol chitosan (MGC) or methacrylated chondroitin sulphate (MCS) delivery vehicles. Stable MGC- and MCS-based composite scaffolds were fabricated containing up to 5 wt% cryomilled DAT through initiation with long-wavelength ultraviolet light. The encapsulation strategy allows for tuning of the 3-D microenvironment and provides an effective method of cell delivery with high seeding efficiency and uniformity, which could be adapted as a minimally-invasive in situ approach. Through in vitro cell culture studies, human ASCs were assessed over 14 days in terms of viability, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, adipogenic gene expression and intracellular lipid accumulation. In all of the composites, the DAT functioned as a cell-supportive matrix that enhanced ASC viability, retention and adipogenesis within the gels. The choice of hydrogel also influenced the cell response, with significantly higher viability and adipogenic differentiation observed in the MCS composites containing 5 wt% DAT. In vivo analysis in a subcutaneous Wistar rat model at 1, 4 and 12 weeks showed superior implant integration and adipogenesis in the MCS-based composites, with allogenic ASCs promoting cell infiltration, angiogenesis and ultimately, fat formation.
An injectable composite scaffold incorporating decellularized adipose tissue (DAT) as a bioactive matrix within a hydrogel phase capable of in situ polymerization would be advantageous for ...adipose-derived stem cell (ASC) delivery in the filling of small or irregular soft tissue defects. Building on previous work, the current study investigates DAT milling methods and the effects of DAT particle size and cell seeding density on the response of human ASCs encapsulated in photo-cross-linkable methacrylated chondroitin sulphate (MCS)-DAT composite hydrogels. DAT particles were generated by milling lyophilized DAT and the particle size was controlled through the processing conditions with the goal of developing composite scaffolds with a tissue-specific 3D microenvironment tuned to enhance adipogenesis. ASC proliferation and adipogenic differentiation were assessed in vitro in scaffolds incorporating small (average diameter of 38 ± 6 μm) or large (average diameter of 278 ± 3 μm) DAT particles in comparison to MCS controls over a period of up to 21 d. Adipogenic differentiation was enhanced in the composites incorporating the smaller DAT particles and seeded at the higher density of 5 × 10(5) ASCs/scaffold, as measured by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, semi-quantitative analysis of perilipin expression and oil red O staining of intracellular lipid accumulation. Overall, this study demonstrates that decellularized tissue particle size can impact stem cell differentiation through cell-cell and cell-matrix interactions, providing relevant insight towards the rational design of composite biomaterial scaffolds for adipose tissue engineering.
Controlled modification of the carboxylic acid moieties of hyaluronic acid with mono- and polyfunctional hydrazides leads to biochemical probes, biopolymers with altered physical and chemical ...properties, tethered drugs for controlled release, and crosslinked hydrogels as biocompatible scaffoldings for tissue engineering. Methods for polyhydrazide synthesis, for prodrug preparation, for hydrogel crosslinking, and for monitoring biodegradation are described.
New polyvalent hydrazide cross-linkers were synthesized, characterized, and used to prepare hydrazide cross-linked hydrogels derived from hyaluronic acid (HA). First, the chemical synthesis and ...characterization of the di-, tri-, tetra-, penta-, and hexahydrazides are presented. Second, HA concentration, buffer type and concentration, and ratio of HA to carbodiimide to cross-linker were varied to obtain HA-hydrogels with different chemical and physical properties. Third, two new assays are described to monitor the stability of HA-hydrogels toward hyaluronidase (HAse) and other media. These assays were used to evaluate the stability of cross-linked HA-hydrogels to HAse solutions and different pH values. Hydrophobic cross-linkers gave the most stable gels, and the susceptibility of the gels to HAse was independent of cross-linker concentration. HAse does not significantly penetrate the HA-hydrogels and acts primarily at the gel−solution interface. The HA-hydrogels are stable in acid environments and dissolve gradually above pH 7.0.
Novel multichannel electrospray emitters are presented that use silica-based microstructured fibers (MSFs) to split the flow allowing efficient desolvation during electrospray. The MSFs investigated ...in this study possess 30−168 individual fluidic channels (each channel being 5 μm in diameter) that form a 2D emitting array. Multiple flow paths afford stable electrospray at flow rates ranging from the microspray (e.g., 1000 nL/min) to the nanoelectrospray (e.g., 10 nL/min) regime with moderate to negligible flow-induced backpressures. The electrospray stability of highly aqueous solutions (up to 99.9% water with 0.1% acetic acid) is enhanced through modification of the emitting surface with a hydrophobic silylation reagent (chlorotrimethylsilane). Furthermore, by successfully spraying highly concentrated salt solutions, this study demonstrates that multichannel MSF emitters provide enhanced robustness to clogging, leading to increased operational throughput.
Surfaces of polypropylene (PP), polystyrene (PS) and polytetrafluoroethylene (PTFE) were activated with radio frequency plasmas Ar and NH
3 to aminate the polymer surface and were subsequently ...reacted with hyaluronic acid (HA) in one of the three different attachment schemes. Results show that ammonia plasma treated polymers were more reactive toward HA attachment. The three chemistry schemes consisted of two distinct approaches: (1) direct attachment of the HA to the aminated surface, and (2) extending the reactive group away from the surface with succinic anhydride and then reacting the newly formed carboxylic acid group with an adipic dihydrazide modified HA (HA-ADH). The latter scheme proved to be more effective, suggesting that steric effects were involved with the reactivity of the HA with surface groups. These HA-coated polymers are a candidate for cell attachment and growth.
Benzophenones (BP) and related aryl ketone photophores have become established as the photoactivatable group of choice for high‐efficiency covalent modification of hydrophobic regions of binding ...proteins, including enzymes and receptors that recognize peptide hormones, (oligonucleotides and nucleosides, phosphoinositides, inositol polyphosphates and a wide variety of therapeutic molecules. This review presents the advantages of BP as pho‐toafnnity labels and provides specific examples from the last 3 years of applications of BP‐containing ligands used in biochemistry.
Novel capillary columns containing polymer-entrapped octadecylsilyl-modified silica microspheres were evaluated for chromatographic performance. The polymer forms only at the particle surface and in ...as little as 10 s strongly immobilizes them, obviating the need for a frit. The photoinitiated polymerization is patternable, and various entrapment styles were compared, all of which withstood pressure drops of at least 5000 psi (345 bar). The fritless nature of the columns allows a unique mass production capability whereby long packed columns are entrapped and simply cut afterward. Since the material has previously been shown to be a proficient nanoelectrospray emitter, the columns have enormous potential for use in capillary LC-MS. The evaluation suggests that for the designs that have minimal entrapped regions, the column performance is equivalent to commercial columns containing similar particles.
Heptaprenyl diphosphate synthase of Bacillus subtilis, which participates in the biosynthesis of the side chain of menaquinone-7, is composed of two dissociable subunits, component I and component ...II, which are encoded by two cistrons in a novel gene cluster of gerC operon Zhang, Y.-W., et al. (1997) J. Bacteriol. 179, 1417-1419. This enzyme essentially requires the coexistence of both subunits for its catalysis. Expression vector systems for the two structural genes, gerC1 and gerC3, were constructed separately, and the two components were overproduced in Escherichia coli cells. After purification, their dynamic interactions in forming a catalytically active complex were investigated by gel filtration and immunoblotting analyses. When a mixture of the two components that had been preincubated in the presence of Mg2+ and farnesyl diphosphate was subjected to Superdex 200 gel filtration, a significant elution peak appeared in a region earlier than those observed when they were chromatographed individually. This fraction contained both components I and II, and it corresponded to a molecular mass that is in accord with the sum of the values of the two components. Cross-linking studies indicate that the two essential subunits, farnesyl diphosphate, and Mg2+ form a ternary complex which seems to represent a catalytically active state of the heptaprenyl diphosphate synthase. On the other hand, no complex was formed in the presence of isopentenyl diphosphate or inorganic pyrophosphate and Mg2+. A photoaffinity analogue of farnesyl diphosphate was shown to preferentially label the component I protein, suggesting that component I possesses a specific affinity for the allylic substrate. Furthermore, the photoaffinity labeling of component I significantly increased in the presence of component II. The mechanism of catalysis of this unique heteromeric enzyme is understood by assuming that association and dissociation of the two subunits facilitate turnover of catalysis for the synthesis of the amphipathic product from soluble substrates.
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