RESUMEN
The macromolecule oligo(poly(ethylene glycol) fumarate) (OPF) exhibits promising attributes for creating suitable three-dimensional hydrogel environments to study cell behavior, deliver therapeutics, and serve as a degradable, nonfouling material. However, traditional synthesis techniques are time consuming, contain salt contaminants, and generate significant waste. These issues have been overcome with an alternative, one-pot approach that utilizes inert gas sparging. Departing from previous synthetic schemes that require acid scavengers, inert gas sparging removes byproducts in situ, eliminating significant filtration and postprocessing steps, while allowing a more uniform product. Characterized by nuclear magnetic resonance, gel permeation chromatography, and differential scanning calorimetry, nitrogen sparge synthesis yields an OPF product with greater polymer length than traditional acid scavenger synthesis methods. Furthermore, nitrogen-sparged OPF readily crosslinks using either ultraviolet or thermal initiator methods with or without the addition of short-chain diacrylate units, allowing for greater tunability in hydrogel properties with little to no cytotoxicity. Overall, inert gas sparging provides a longer chain and cleaner polymer product for hydrogel material studies while maintaining degradable characteristics. Impact statement Using nitrogen sparging, we have demonstrated that oligo(poly(ethylene glycol) fumarate) (OPF) can be produced with decreased postprocessing, increased product purity, greater oligomerization, and cell viability. These properties lead to greater tunability in mechanical properties and a more versatile hydrogel for biomedical applications. The simplification of synthesis and elimination of impurities will expand the utility of OPF as a degradable hydrogel for cell culture, tissue engineering, regenerative medicine, and therapeutic delivery, among other applications.
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Hidrogeles , Polietilenglicoles , Supervivencia Celular , Fumaratos , Ingeniería de TejidosRESUMEN
BACKGROUND: Yersinia pestis is a category A infective agent that causes bubonic, septicemic, and pneumonic plague. Notably, the acquisition of antimicrobial or multidrug resistance through natural or purposed means qualifies Y. pestis as a potential biothreat agent. Therefore, high-quality antibodies designed for accurate and sensitive Y. pestis diagnostics, and therapeutics potentiating or replacing traditional antibiotics are of utmost need for national security and public health preparedness. METHODS: Here, we describe a set of human monoclonal immunoglobulins (IgG1s) targeting Y. pestis fraction 1 (F1) antigen, previously derived from in vitro evolution of a phage-display library of single-chain antibodies (scFv). We extensively characterized these antibodies and their effect on bacterial and mammalian cells via: ELISA, flow cytometry, mass spectrometry, spectroscopy, and various metabolic assays. RESULTS: Two of our anti-F1 IgG (αF1Ig 2 and αF1Ig 8) stood out for high production yield, specificity, and stability. These two antibodies were additionally attractive in that they displayed picomolar affinity, did not compete when binding Y. pestis, and retained immunoreactivity upon chemical derivatization. Most importantly, these antibodies detected <1,000 Y. pestis cells in sandwich ELISA, did not harm respiratory epithelial cells, induced Y. pestis agglutination at low concentration (350 nM), and caused apparent reduction in cell growth when radiolabeled at a nonagglutinating concentration (34 nM). CONCLUSION: These antibodies are amenable to the development of accurate and sensitive diagnostics and immuno/radioimmunotherapeutics.
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Previous methods for investigating material stiffness on cell behavior have focused on the use of substrates with limited ranges of stiffness and/or fluctuating surface chemistries. Using the co-polymer system of n-octyl methacrylate crosslinked with diethylene glycol dimethacrylate (DEGDMA/nOM), we developed a new cell culture platform to analyze the isolated effects of stiffness independent from changes in surface chemistry. Materials ranging from 25 kPa to 4,700 kPa were fabricated. Surface analysis including goiniometry and X-ray photoelectron spectroscopy (XPS) confirmed consistent surface chemistry across all formulations examined. The mechanosensitive cell type valvular interstitial cell (VIC) was cultured DEGDMA/nOM substrates of differing stiffness. Results indicate that order of magnitude changes in stiffness do not increase gene expression of VIC alpha-smooth muscle actin (αSMA). However, structural organization of αSMA is altered on stiffer substrates, corresponding with the appearance of the osteoblastic marker osteocalcin and nodule formation. This research presents the co-polymer DEGDMA/nOM as ideal substrate to investigate the influence of stiffness on VIC differentiation without the confounding effects of changing material surface chemistry. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 51-61, 2017.
Asunto(s)
Antígenos de Diferenciación/biosíntesis , Diferenciación Celular , Válvulas Cardíacas/metabolismo , Metacrilatos/química , Osteoblastos/metabolismo , Animales , Células Cultivadas , Válvulas Cardíacas/citología , Osteoblastos/citología , PorcinosRESUMEN
The primary driver for valvular calcification is the differentiation of valvular interstitial cells (VICs) into a diseased phenotype. However, the factors leading to the onset of osteoblastic-like VICs (obVICs) and resulting calcification are not fully understood. This study isolates the effect of substrate surface chemistry on in vitro VIC differentiation and calcified tissue formation. Using ω-functionalized alkanethiol self-assembled monolayers (SAMs) on gold [CH3 (hydrophobic), OH (hydrophilic), COOH (COO(-), negative at physiological pH), and NH2 (NH3(+), positive at physiological pH)], we have demonstrated that surface chemistry modulates VIC phenotype and calcified tissue deposition independent of osteoblastic-inducing media additives. Over seven days VICs exhibited surface-dependent differences in cell proliferation (COO(-)=NH3(+)>OH>CH3), morphology, and osteoblastic potential. Both NH3(+)and CH3-terminated SAMs promoted calcified tissue formation while COO(-)-terminated SAMs showed no calcification. VICs on NH3(+)-SAMs exhibited the most osteoblastic phenotypic markers through robust nodule formation, up-regulated osteocalcin and α-smooth muscle actin expression, and adoption of a round/rhomboid morphology indicative of osteoblastic differentiation. With the slowest proliferation, VICs on CH3-SAMs promoted calcified aggregate formation through cell detachment and increased cell death indicative of dystrophic calcification. Furthermore, induction of calcified tissue deposition on NH3(+) and CH3-SAMs was distinctly different than that of media induced osteoblastic VICs. These results demonstrate that substrate surface chemistry alters VIC behavior and plays an important role in calcified tissue formation. In addition, we have identified two novel methods of calcified VIC induction in vitro. Further study of these environments may yield new models for in vitro testing of therapeutics for calcified valve stenosis, although additional studies need to be conducted to correlate results to in vivo models. STATEMENT OF SIGNIFICANCE: Valvular interstitial cell (VIC) differentiation and aortic valve calcification is associated with increased risk of mortality and onset of other cardiovascular disorders. This research examines effects of in vitro substrate surface chemistry on VIC differentiation and has led to the identification of two materials-based initiation mechanisms of osteoblastic-like calcified tissue formation independent of soluble signaling methods. Such findings are important for their potential to study signaling cascades responsible for valvular heart disease initiation and progression as well providing in vitro disease models for drug development. We have also identified a VIC activating in vitro environment that does not exhibit confluence induced nodule formation with promise for the development of tissue regenerating scaffolds.