Cell Attachment and Spreading on Carbon Nanotubes Is Facilitated by Integrin Binding

Owing to their exceptional physical, chemical, and mechanical properties, carbon nanotubes (CNTs) have been extensively studied for their effect on cellular behaviors. However, little is known about the process by which cells attach and spread on CNTs and the process for cell attachment and spreading on individual single-walled CNTs has not been studied. Cell adhesion and spreading is essential for cell communication and regulation and the mechanical interaction between cells and the underlying substrate can influence and control cell behavior and function. A limited number of studies have described different adhesion mechanisms, such as cellular process entanglements with multi-walled CNT aggregates or adhesion due to adsorption of serum proteins onto the nanotubes. Here, we hypothesized that cell attachment and spreading to both individual single-walled CNTs and multi-walled CNT aggregates is governed by the same mechanism. Specifically, we suggest that cell attachment and spreading on nanotubes is integrin-dependent and is facilitated by the adsorption of serum and cell-secreted adhesive proteins to the nanotubes

Star poly(ethylene glycol) as a tunable scaffold for neural tissue engineering

The primary focus of this work was to develop a novel synthetic hydrogel scaffold as an in vitro model to enable future detailed studies of how neurons grow in environments with controllable diffusion profiles of soluble cues and tunable neuron-matrix interactions. The development of in vitro models that enable elucidation of the mechanisms of system performance is a recently emerging goal of tissue engineering. The design of three-dimensional (3D) scaffolds in particular, is motivated by the need to develop model systems that better mimic native tissue as compared to conventional two-dimensional (2D) cell culture substrates. An ideal scaffold is degradable, porous, biocompatible, with mechanical properties to match those of the tissues of interest and with a suitable surface chemistry for cell attachment, proliferation, and differentiation. Although naturally derived materials are more versatile in providing complex biological cues, synthetic polymers are preferable for the design of in vitro models as they provide wider range of properties, controllable degradation rates, and easier processing. Most importantly, their mechanical properties can be decoupled from their biological properties, a crucial issue in interpreting cell responses. The synthetic material provides the structural backbone of the scaffold while biochemical function is added via incorporation of ligands or proteins aimed at triggering specific cell behaviors. As presented in this dissertation, we have developed and characterized a new synthetic 3D hydrogel scaffold from cross-linked poly(ethylene glycol) (PEG). PEG was selected because it is hydrophilic, non-toxic, biocompatible, and inert to protein adhesion. The chosen cross-linking chemistry was a highly specific reaction that occurred under physiological conditions so that cells could be embedded within the gel prior to cross-linking. Controllable degradability was imparted via series of hydrolytically degradable PEG cross-linkers. Thorough analysis demonstrated the independent tuning of the mechanical, biochemical and biological properties of the developed hydrogel. Because soluble cues such as neurotrophic factors are an effective means for promoting nerve regeneration, the diffusion of biomolecules through the PEG hydrogel were also explored in depth via two methods: fluorescence correlation spectroscopy (FCS) and bulk diffusion experiments. This is the first demonstration of FCS to delineate protein diffusivity within a cross-linked synthetic hydrogel and describe local and dynamic protein-polymer interactions that occur within these systems. Further, since PEG is inert, short ligands such as RGD were used to promote cell adhesion and new insights into how these ligands impact hydrogel mechanical and transport properties were established. Finally, to test the utility of the developed material as an in vitro model, neuronal cell-matrix interactions were studied by tuning hydrogel properties and assessing cell viability and neurite outgrowth. We believe that this work is major step in building an in vitro model for gaining an understanding of the key parameters that guide nerve regeneration and have the potential to lead to the development of better strategies to treat peripheral nerve injuries

Star poly(ethylene glycol) as a tunable scaffold for neural tissue engineering

The primary focus of this work was to develop a novel synthetic hydrogel scaffold as an in vitro model to enable future detailed studies of how neurons grow in environments with controllable diffusion profiles of soluble cues and tunable neuron-matrix interactions. The development of in vitro models that enable elucidation of the mechanisms of system performance is a recently emerging goal of tissue engineering. The design of three-dimensional (3D) scaffolds in particular, is motivated by the need to develop model systems that better mimic native tissue as compared to conventional two-dimensional (2D) cell culture substrates. An ideal scaffold is degradable, porous, biocompatible, with mechanical properties to match those of the tissues of interest and with a suitable surface chemistry for cell attachment, proliferation, and differentiation. Although naturally derived materials are more versatile in providing complex biological cues, synthetic polymers are preferable for the design of in vitro models as they provide wider range of properties, controllable degradation rates, and easier processing. Most importantly, their mechanical properties can be decoupled from their biological properties, a crucial issue in interpreting cell responses. The synthetic material provides the structural backbone of the scaffold while biochemical function is added via incorporation of ligands or proteins aimed at triggering specific cell behaviors. As presented in this dissertation, we have developed and characterized a new synthetic 3D hydrogel scaffold from cross-linked poly(ethylene glycol) (PEG). PEG was selected because it is hydrophilic, non-toxic, biocompatible, and inert to protein adhesion. The chosen cross-linking chemistry was a highly specific reaction that occurred under physiological conditions so that cells could be embedded within the gel prior to cross-linking. Controllable degradability was imparted via series of hydrolytically degradable PEG cross-linkers. Thorough analysis demonstrated the independent tuning of the mechanical, biochemical and biological properties of the developed hydrogel. Because soluble cues such as neurotrophic factors are an effective means for promoting nerve regeneration, the diffusion of biomolecules through the PEG hydrogel were also explored in depth via two methods: fluorescence correlation spectroscopy (FCS) and bulk diffusion experiments. This is the first demonstration of FCS to delineate protein diffusivity within a cross-linked synthetic hydrogel and describe local and dynamic protein-polymer interactions that occur within these systems. Further, since PEG is inert, short ligands such as RGD were used to promote cell adhesion and new insights into how these ligands impact hydrogel mechanical and transport properties were established. Finally, to test the utility of the developed material as an in vitro model, neuronal cell-matrix interactions were studied by tuning hydrogel properties and assessing cell viability and neurite outgrowth. We believe that this work is major step in building an in vitro model for gaining an understanding of the key parameters that guide nerve regeneration and have the potential to lead to the development of better strategies to treat peripheral nerve injuries

Adsorption and Sustained Delivery of Small Molecules from Nanosilicate Hydrogel Composites

Two-dimensional nanosilicate particles (NS) have shown promise for the prolonged release of small-molecule therapeutics while minimizing burst release. When incorporated in a hydrogel, the high surface area and charge of NS enable electrostatic adsorption and/or intercalation of therapeutics, providing a lever to localize and control release. However, little is known about the physio-chemical interplay between the hydrogel, NS, and encapsulated small molecules. Here, we fabricated polyethylene glycol (PEG)-NS hydrogels for the release of model small molecules such as acridine orange (AO). We then elucidated the effect of NS concentration, NS/AO incubation time, and the ability of NS to freely associate with AO on hydrogel properties and AO release profiles. Overall, NS incorporation increased the hydrogel stiffness and decreased swelling and mesh size. When individual NS particles were embedded within the hydrogel, a 70-fold decrease in AO release was observed compared to PEG-only hydrogels, due to adsorption of AO onto NS surfaces. When NS was pre-incubated and complexed with AO prior to hydrogel encapsulation, a >9000-fold decrease in AO release was observed due to intercalation of AO between NS layers. Similar results were observed for other small molecules. Our results show the potential for use of these nanocomposite hydrogels for the tunable, long-term release of small molecules

Homogenization Theory for the Prediction of Obstructed Solute Diffusivity in Macromolecular Solutions.

The study of diffusion in macromolecular solutions is important in many biomedical applications such as separations, drug delivery, and cell encapsulation, and key for many biological processes such as protein assembly and interstitial transport. Not surprisingly, multiple models for the a-priori prediction of diffusion in macromolecular environments have been proposed. However, most models include parameters that are not readily measurable, are specific to the polymer-solute-solvent system, or are fitted and do not have a physical meaning. Here, for the first time, we develop a homogenization theory framework for the prediction of effective solute diffusivity in macromolecular environments based on physical parameters that are easily measurable and not specific to the macromolecule-solute-solvent system. Homogenization theory is useful for situations where knowledge of fine-scale parameters is used to predict bulk system behavior. As a first approximation, we focus on a model where the solute is subjected to obstructed diffusion via stationary spherical obstacles. We find that the homogenization theory results agree well with computationally more expensive Monte Carlo simulations. Moreover, the homogenization theory agrees with effective diffusivities of a solute in dilute and semi-dilute polymer solutions measured using fluorescence correlation spectroscopy. Lastly, we provide a mathematical formula for the effective diffusivity in terms of a non-dimensional and easily measurable geometric system parameter

Laponite-Based Nanocomposite Hydrogels for Drug Delivery Applications

Hydrogels are widely used for therapeutic delivery applications due to their biocompatibility, biodegradability, and ability to control release kinetics by tuning swelling and mechanical properties. However, their clinical utility is hampered by unfavorable pharmacokinetic properties, including high initial burst release and difficulty in achieving prolonged release, especially for small molecules (<500 Da). The incorporation of nanomaterials within hydrogels has emerged as viable option as a method to trap therapeutics within the hydrogel and sustain release kinetics. Specifically, two-dimensional nanosilicate particles offer a plethora of beneficial characteristics, including dually charged surfaces, degradability, and enhanced mechanical properties within hydrogels. The nanosilicate–hydrogel composite system offers benefits not obtainable by just one component, highlighting the need for detail characterization of these nanocomposite hydrogels. This review focuses on Laponite, a disc-shaped nanosilicate with diameter of 30 nm and thickness of 1 nm. The benefits of using Laponite within hydrogels are explored, as well as examples of Laponite–hydrogel composites currently being investigated for their ability to prolong the release of small molecules and macromolecules such as proteins. Future work will further characterize the interplay between nanosilicates, hydrogel polymer, and encapsulated therapeutics, and how each of these components affect release kinetics and mechanical properties

Morphological adaptations in breast cancer cells as a function of prolonged passaging on compliant substrates

<div><p>Standard tissue culture practices involve propagating cells on tissue culture polystyrene (TCP) dishes, which are flat, 2-dimensional (2D) and orders of magnitude stiffer than most tissues in the body. Such simplified conditions lead to phenotypical cell changes and altered cell behaviors. Hence, much research has been focused on developing novel biomaterials and culture conditions that more closely emulate <i>in vivo</i> cell microenvironments. In particular, biomaterial stiffness has emerged as a key property that greatly affects cell behaviors such as adhesion, morphology, proliferation and motility among others. Here we ask whether cells that have been conditioned to TCP, would still show significant dependence on substrate stiffness if they are first pre-adapted to a more physiologically relevant environment. We used two commonly utilized breast cancer cell lines, namely MDA-MB-231 and MCF-7, and examined the effect of prolonged cell culturing on polyacrylamide substrates of varying compliance. We followed changes in cell adhesion, proliferation, shape factor, spreading area and spreading rate. After pre-adaptation, we noted diminished differences in cell behaviors when comparing between soft (1 kPa) and stiff (103 kPa) gels as well as rigid TCP control. Prolonged culturing of cells on complaint substrates further influenced responses of pre-adapted cells when transferred back to TCP. Our results have implications for the study of stiffness-dependent cell behaviors and indicate that cell pre-adaptation to the substrate needs consideration.</p></div

Carrageenan-Based Crowding and Confinement Combination Approach to Increase Collagen Deposition for In Vitro Tissue Development

Connective tissue models grown from cell monolayers can be instrumental in a variety of biomedical fields such as drug screening, wound healing, and regenerative engineering. However, while connective tissues contain abundant fibrillar collagen, achieving a sufficient assembly and retention of fibrillar collagen in vitro is challenging. Unlike the dilute cell culture environment, the body’s environment is characterized by a high density of soluble macromolecules (crowding) and macromolecular networks (confinement), which contribute to extracellular matrix (ECM) assembly in vivo. Consequently, macromolecular crowding (MMC) has been successfully used to enhance the processing of type I procollagen, leading to significant increases in fibrillar collagen assembly and accumulation during in vitro culture of a variety of cell types. In this study, we developed a combination approach using a carrageenan hydrogel, which released soluble macromolecules and served as a confinement barrier. We first evaluated the local carrageenan release and then confirmed the effectiveness of this combination approach on collagen accumulation by the human MG-63 bone cell line. Additionally, computational modeling of oxygen and glucose transport within the culture system showed no negative effects of the hydrogel and its releasates on cell viability

Histograms of MDA-MB-231 cell spreading area and circularity expressed as percent occurrence.

<p>Spreading area and circularity were measured at 72 h of each passage (P1, P2 and P3). (A) Percent occurrence of cell spreading area and circularity on TCP. (B) Percent occurrence of cell spreading area and (C) circularity on 103 kPa gels. (D) Percent occurrence of cell spreading area and (E) circularity on 1 kPa gels.</p

Cell spreading area of MDA-MB-231 cells.

<p>Cell spreading area was measured after cells were transferred onto TCP from 3 difference’ conditions: TCP control (TCP→TCP), P3 on 103 kPa gel (103→TCP), and P3 on 1 kPa gel (1→TCP). *represent significant difference from 24 h, ^#represents significant difference from TCP→TCP (p<0.05, n = 3).</p