Aaron M. Scurto

Microsphere-Based Scaffolds for Cartilage Tissue Engineering: Using Sub-critical CO2 as a Sintering Agent

Shape-specific, macroporous tissue engineering scaffolds were fabricated and homogeneously seeded with cells in a single step. This method brings together CO(2) polymer processing and microparticle-based scaffolds in a manner that allows each to solve the key limitation of the other. Specifically, microparticle-based scaffolds have suffered from the limitation that conventional microsphere sintering methods (e.g., heat, solvents) are not cytocompatible, yet we have shown that cell viability was sustained with subcritical (i.e., gaseous) CO(2) sintering of microspheres in the presence of cells at near-ambient temperatures. On the other hand, the fused microspheres provided the pore interconnectivity that has eluded supercritical CO(2) foaming approaches. Here, fused poly(lactide-co-glycolide) microsphere scaffolds were seeded with human umbilical cord mesenchymal stromal cells to demonstrate the feasibility of utilizing these matrices for cartilage regeneration. We also demonstrated that the approach may be modified to produce thin cell-loaded patches as a promising alternative for skin tissue engineering applications.

Carbon dioxide induced separation of ionic liquids and water

Both hydrophobic and hydrophilic room-temperature ionic liquids can be separated from aqueous solutions with relatively low-pressure gaseous carbon dioxide.

Design of ionic liquids via computational molecular design

Abstract Computational molecular design (CMD) is a methodology which applies optimization techniques to develop novel lead compounds for a variety of applications. In this work, a CMD method is applied to the design of ionic liquids (ILs), which are being considered for use as environmentally benign solvents. The molecularly tunable nature of ILs yields an extraordinary number of possible cation and anion combinations, the majority of which have never been synthesized. The product design framework developed in this work seeks to accelerate the commonly used experimental trial-and-error approach by searching through this large molecular space and providing a set of chemical structures likely to match a set of desired property targets. To predict the physical and chemical properties of an ionic liquid in a specific system, quantitative structure–property relations (QSPRs) have been developed. In this work, correlations were created for solubility, diffusivity, and melting temperature. The electronic structure of ionic liquids is quantified using molecular connectivity indices, which describe bonding environments, charge distribution, orbital hybridization and other interactions within and between ions. The resulting property prediction model is then integrated within a computational molecular design framework, which combines the QSPRs with structural feasibility constraints in a combinatorial optimization problem. The problem is reformulated as an MILP after exact linearization of structural constraints. An example is provided to test the formulation for the design of ionic liquids for use within a hydrofluorocarbon (refrigerant) gas separation system. A second example compares a stochastic optimization algorithm, Tabu Search, to a standard deterministic solver for the solution of a larger-scale refrigeration design problem. The computational efficiency and practical implementation of this product design methodology is also discussed.

High-pressure phase equilibria with compressed gases

An apparatus is described that is capable of determining high-pressure vapor-liquid equilibrium, liquid-liquid equilibrium, solid-liquid-vapor equilibrium, vapor-liquid-liquid equilibrium, and mixture critical points and transitions. The device is capable of temperatures to 150 degrees C and pressures to 300 bars (higher with slight modifications). The construction and operation are described in detail and do not require the use of mercury. This method requires very low sample volumes and no analytical equipment nor system-specific calibration. The apparatus was verified by comparison with literature data for the decane-CO(2) mixture and CO(2)-ionic liquid [1-hexyl-3-methyl-imidazolium bis(trifyl)imide)] systems. The experimental data have excellent agreement with the literature data that used different experimental methods. A rigorous error analysis of the system is also presented.

Cellulose Solubility in Ionic Liquid Mixtures: Temperature, Cosolvent, and Antisolvent Effects

Select ionic liquids (ILs) dissolve significant quantities of cellulose through disruption and solvation of inter- and intramolecular hydrogen bonds. In this study, thermodynamic solid-liquid equilibrium was measured with microcrystalline cellulose in a model IL, 1-ethyl-3-methylimidazolium diethyl phosphate ([EMIm][DEP]) and mixtures with protic antisolvents and aprotic cosolvents between 40 and 120 °C. The solubility of cellulose in pure [EMIm][DEP] exhibits an asymptotic maximum of approximately 20 mass % above 100 °C. Solubility studies conducted on antisolvent mixtures with [EMIm][DEP] and [BMIm][Cl] indicate that protic solvents, ethanol, methanol, and water, significantly reduce the cellulose capacity of IL mixtures by 38-100% even at small antisolvent loadings (<5 mass %). Alternatively, IL-aprotic cosolvent (dimethyl sulfoxide, dimethylformamide, and 1,3-dimethyl-2-imidazolidinone) mixtures at mass ratios up to 1:1 enhance cellulose dissolution by 20-60% compared to pure [EMIm][DEP] at select temperatures. Interactions between the IL and molecular solvents were investigated by Kamlet-Taft solvatochromic analysis, FTIR, and NMR spectroscopy. The results indicate that preferential solvation of the IL cation and anion by co- and antisolvents impact the ability of IL ions to interact with cellulose thus affecting the cellulose dissolution capacity of IL-solvent mixtures.

Kinetics and solvent effects in the synthesis of ionic liquids: imidazolium

Ionic liquids (ILs) are being considered as a promising class of potentially environmentally-friendly (“green”) solvents and materials for use in a variety of applications. However, ionic liquids are conventionally synthesized by batch, without known kinetics, in non-sustainable solvents. For ILs to be a truly “green” technology for widespread use, they must themselves be made in a correspondingly benign manner for low cost, as enabled by process development. This investigation will illustrate the kinetics and large solvent effects in the synthesis of 1-hexyl-3-methyl-imidazolium bromide in 10 different solvents: acetone, acetonitrile, 2-butanone, chlorobenzene, dichloromethane, dimethyl sulfoxide (DMSO), ethyl formate, ethyl lactate, methanol, and cyclopentanone. The kinetic rate constant for the synthesis in DMSO is over an order-of-magnitude larger than that in methanol. While the kinetic rate of these type of SN2 reactions is generally known to increase with solvent “polarity”, multi-parameter solvent descriptors, e.g. of Kamlet and Taft, are required to quantify these effects in a Linear Solvation Energy Relationship. These relationships are used with environmental and toxicity databases, such as the Rowan Solvent Selection Table, to rapidly optimize the solvent for favorable kinetics and minimal human and environmental impact.

The Future of Carbon Dioxide for Polymer Processing in Tissue Engineering

The use of CO2 for scaffold fabrication in tissue engineering was popularized in the mid-1990 s as a tool for producing polymeric foam scaffolds, but had fallen out of favor to some extent, in part due to challenges with pore interconnectivity. Pore interconnectivity issues have since been resolved by numerous dedicated studies that have collectively outlined how to control the appropriate parameters to achieve a pore structure desirable for tissue regeneration. In addition to CO2 foaming, several groups have leveraged CO2 as a swelling agent to impregnate scaffolds with drugs and other bioactive additives, and for encapsulation of plasmids within scaffolds for gene delivery. Moreover, in contrast to CO2 foaming, which typically relies on supercritical CO2 at very high pressures, CO2 at much lower pressures has also been used to sinter polymeric microspheres together in the presence of cells to create cell-seeded scaffolds in a single step. CO2 has a number of advantages for polymer processing in tissue engineering, including its ease of use, low cost, and the opportunity to circumvent the use of organic solvents. Building on these advantages, and especially now with the tremendous precedent that has paved the way in defining operating parameters, and making the technology accessible for new groups to adapt, we invite and encourage our colleagues in the field to leverage CO2 as a new tool to enhance their own respective unique capabilities.

Effect of different sintering methods on bioactivity and release of proteins from PLGA microspheres

Macromolecule release from poly(d,l-lactide-co-glycolide) (PLGA) microspheres has been well-characterized, and is a popular approach for delivering bioactive signals from tissue-engineered scaffolds. However, the effect of some processing solvents, sterilization, and mineral incorporation (when used in concert) on long-term release and bioactivity has seldom been addressed. Understanding these effects is of significant importance for microsphere-based scaffolds, given that these scaffolds are becoming increasingly more popular, yet growth factor activity following sintering and/or sterilization is heretofore unknown. The current study evaluated the 6-week release of transforming growth factor (TGF)-β3 and bone morphogenetic protein (BMP)-2 from PLGA and PLGA/hydroxyapatite (HAp) microspheres following exposure to ethanol (EtOH), dense phase carbon dioxide (CO2), or ethylene oxide (EtO). EtO was chosen based on its common use in scaffold sterilization, whereas EtOH and CO2 were chosen given their importance in sintering microspheres together to create scaffolds. Release supernatants were then used in an accelerated cell stimulation study with human bone marrow stromal cells (hBMSCs) with monitoring of gene expression for major chondrogenic and osteogenic markers. Results indicated that in microspheres without HAp, EtOH exposure led to the greatest amount of delivery, while those treated with CO2 delivered the least growth factor. In contrast, formulations with HAp released almost half as much protein, regardless of EtOH or CO2 exposure. Notably, EtO exposure was not found to significantly affect the amount of protein released. Cell stimulation studies demonstrated that eluted protein samples performed similarly to positive controls in PLGA-only formulations, and ambiguously in PLGA/HAp composites. In conclusion, the use of EtOH, subcritical CO2, and EtO in microsphere-based scaffolds may have only slight adverse effects, and possibly even desirable effects in some cases, on protein availability and bioactivity.

Carbon Dioxide Solubility Enhancement through Silicone Functionalization: “CO2-philic” Oligo(dimethylsiloxane)-substituted Diphosphonates∗

Abstract Carbon dioxide has received significant attention as a potential environmentally benign medium to replace hazardous organic compounds, but is a relatively poor solvent. The addition of siloxane substituents provides an attractive and inexpensive means to solubilize a wide variety of compounds in CO2. By synthesizing and testing a family of gem-diphosphonate ligands that have been rendered CO2-philic by incorporation of a number of related, discrete dimethylsiloxane oligomers, we show that small variations in substituents have a significant effect on the CO2-philicity of the ligand. To our knowledge, this is the first systematic study of the effect of siloxane substituent size, branching, and position on the affinity of a ligand for CO2. In addition, we present a general approach to the preparation of novel gem-diphosphonate ligands.

Subcritical CO2 sintering of microspheres of different polymeric materials to fabricate scaffolds for tissue engineering.

The aim of this study was to use CO2 at sub-critical pressures as a tool to sinter 3D, macroporous, microsphere-based scaffolds for bone and cartilage tissue engineering. Porous scaffolds composed of ~200 μm microspheres of either poly(lactic-co-glycolic acid) (PLGA) or polycaprolactone (PCL) were prepared using dense phase CO2 sintering, which were seeded with rat bone marrow mesenchymal stromal cells (rBMSCs), and exposed to either osteogenic (PLGA, PCL) or chondrogenic (PLGA) conditions for 6 weeks. Under osteogenic conditions, the PLGA constructs produced over an order of magnitude more calcium than the PCL constructs, whereas the PCL constructs had far superior mechanical and structural integrity (125 times stiffer than PLGA constructs) at week 6, along with twice the cell content of the PLGA constructs. Chondrogenic cell performance was limited in PLGA constructs, perhaps as a result of the polymer degradation rate being too high. The current study represents the first long-term culture of CO2-sintered microsphere-based scaffolds, and has established important thermodynamic differences in sintering between the selected formulations of PLGA and PCL, with the former requiring adjustment of pressure only, and the latter requiring the adjustment of both pressure and temperature. Based on more straightforward sintering conditions and more favorable cell performance, PLGA may be the material of choice for microspheres in a CO2 sintering application, although a different PLGA formulation with the encapsulation of growth factors, extracellular matrix-derived nanoparticles, and/or buffers in the microspheres may be advantageous for achieving a more superior cell performance than observed here.