Acid Scavenger Free Synthesis of Oligo(Poly(Ethylene Glycol) Fumarate) Utilizing Inert Gas Sparging.

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.

Optimization of electrospun poly(N-isopropyl acrylamide) mats for the rapid reversible adhesion of mammalian cells.

Poly(N-isopropyl acrylamide) (pNIPAM) is a "smart" polymer that responds to changes in altering temperature near physiologically relevant temperatures, changing its relative hydrophobicity. Mammalian cells attach to pNIPAM at 37 °C and detach spontaneously as a confluent sheet when the temperature is shifted below the lower critical solution temperature (∼32 °C). A variety of methods have been used to create pNIPAM films, including plasma polymerization, self-assembled monolayers, and electron beam ionization. However, detachment of confluent cell sheets from these pNIPAM films can take well over an hour to achieve potentially impacting cellular behavior. In this work, pNIPAM mats were prepared via electrospinning (i.e., espNIPAM) by a previously described technique that the authors optimized for cell attachment and rapid cell detachment. Several electrospinning parameters were varied (needle gauge, collection time, and molecular weight of the polymer) to determine the optimum parameters. The espNIPAM mats were then characterized using Fourier-transform infrared, x-ray photoelectron spectroscopy, and scanning electron microscopy. The espNIPAM mats showing the most promise were seeded with mammalian cells from standard cell lines (MC3T3-E1) as well as cancerous tumor (EMT6) cells. Once confluent, the temperature of the cells and mats was changed to ∼25 °C, resulting in the extremely rapid swelling of the mats. The authors find that espNIPAM mats fabricated using small, dense fibers made of high molecular weight pNIPAM are extremely well-suited as a rapid release method for cell sheet harvesting.

Isolated effect of material stiffness on valvular interstitial cell differentiation.

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.

Surface chemistry regulates valvular interstitial cell differentiation in vitro.

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.

Porous poly(DL-lactic-co-glycolic acid)/calcium phosphate cement composite for reconstruction of bone defects.

Calcium phosphate (Ca-P) cements are injectable, self-setting ceramic pastes generally known for their favorable bone response. Ingrowth of bone and subsequent degradation rates can be enhanced by the inclusion of macropores. Initial porosity can be induced by CO(2) foaming during setting of the cement, whereas secondary porosity can develop after hydrolysis of incorporated poly(DL-lactic- co-glycolic acid) (PLGA) microparticles. In this study, we focused on the biological response to porous PLGA/Ca-P cement composites. Pre-set composite discs of four formulations (4 wt% or 15 wt% PLGA microparticles and low or high CO(2) induced porosity) were implanted subcutaneously and in cranial defects in rats for 12 weeks. Histological analysis of the explanted composites revealed that bone and fibrous tissue ingrowth was facilitated by addition of PLGA microparticles (number average diameter of 66 +/- 25 microm). No adverse tissue reaction was observed in any of the composites. Significant increases in composite density due to bone ingrowth in cranial implants were found in all formulations. The results suggest that the PLGA pores are suitable for bone ingrowth and may be sufficient to enable complete tissue ingrowth without initial CO(2) induced porosity. Finally, bone-like mineralization in subcutaneous implants suggests that, under appropriate conditions and architecture, porous PLGA/Ca-P cement composites can exhibit osteoinductive properties. These PLGA/Ca-P composites are a promising scaffolding material for bone regeneration and bone tissue engineering.