Silviya P. Zustiak

Characterization of Protein Release from Hydrolytically Degradable Poly(ethylene glycol) Hydrogels

We present a novel fully hydrophilic, hydrolytically degradable poly(ethylene glycol) (PEG) hydrogel suitable for soft tissue engineering and delivery of protein drugs. The gels were designed to overcome drawbacks associated with current PEG hydrogels (i.e., reaction mechanisms or degradation products that compromise protein stability): the highly selective and mild cross‐linking reaction allowed for encapsulating proteins prior to gelation without altering their secondary structure as shown by circular dichroism experiments. Further, hydrogel degradation and structure, represented by mesh size, were correlated to protein release. It was determined that polymer density had the most profound effect on protein diffusivity, followed by the polymer molecular weight, and finally by the specific chemical structure of the cross‐linker. By examining the diffusion of several model proteins, we confirmed that the protein diffusivity was dependent on protein size as smaller proteins (e.g., lysozyme) diffused faster than larger proteins (e.g., Ig). Furthermore, we demonstrated that the protein physical state was preserved upon encapsulation and subsequent release from the PEG hydrogels and contained negligible aggregation or protein–polymer adducts. These initial studies indicate that the developed PEG hydrogels are suitable for release of stable proteins in drug delivery and tissue engineering applications. Biotechnol. Bioeng. 2011; 108:197–206.

Influence of Cell-Adhesive Peptide Ligands on Poly(ethylene glycol) Hydrogel Physical, Mechanical and Transport Properties

Synthetic three-dimensional scaffolds for cell and tissue engineering routinely utilize peptide ligands to provide sites for cell adhesion and to promote cellular activity. Given the fact that recent studies have dedicated great attention to the mechanisms by which cell behavior is influenced by various ligands and scaffold material properties, it is surprising that little work to date has been carried out to investigate the influence of covalently bound ligands on hydrogel material properties. Herein we report the influence of three common ligands utilized in tissue engineering, namely RGD, YIGSR and IKVAV, on the mechanical properties of cross-linked poly(ethylene glycol) (PEG) hydrogels. The effect of the ligands on hydrogel storage modulus, swelling ratio, mesh size and also on the diffusivity of bovine serum albumin through the hydrogel were investigated in detail. We identified conditions under which these ligands strikingly influence the properties of the material. The extent of influence and whether the ligand increases or decreases a specific property is linked to ligand type and concentration. Further, we pinpoint mechanisms by which the ligands interact with the PEG network. This work thus provides specific evidence for interactions between peptide ligands and cross-linked PEG hydrogels that have a significant impact on hydrogel material and transport properties. As a result, this work may have important implications for interpreting cell experiments carried out with ligand-modified hydrogels, because the addition of ligand may affect not only the scaffolds biological properties, but also key physical properties of the system.

Solute diffusion and interactions in cross-linked poly(ethylene glycol) hydrogels studied by Fluorescence Correlation Spectroscopy

Controlled diffusion and release of soluble molecules is one of the key challenges in developing three-dimensional (3D) scaffolds for tissue engineering and drug delivery applications in part because current methods to measure dynamic transport properties are difficult to perform directly, are strongly affected by the experimental setup, and therefore can be a subject to various artifacts. In this work we present a method for direct measurement of translational diffusion of solutes, namely Fluorescence Correlation Spectroscopy (FCS), by characterizing the diffusion of model proteins through a 3D cross-linked poly(ethylene glycol) (PEG) hydrogel scaffold. We examined both the dynamics of hydrogel structure (e.g., cross-linking and swelling) as well as protein size and their effect on protein diffusivity. For example, we demonstrated that protein diffusivity was closely related to protein size as smaller proteins (e.g., lysozyme) diffused faster than larger proteins (e.g., γ-globulin or Ig). We validated the FCS protein diffusivity results by comparison to standard bulk diffusion assays. Additionally, due to the nature of FCS measurements, we were able to probe for hydrogel-protein interactions during cross-linking that may contribute to the obstructed protein diffusion in the 3D scaffold. We determined that such interactions in this system were not covalent (i.e., were independent of the cross-linking chemistry) but may be due to weaker hydrogen bonding or ionic interactions. Also, these interactions were protein specific and contributed up to 25% of the total decrease in protein diffusivity in the hydrogel as compared to diffusivity in water. Though interactions between various proteins and PEG have been reported, this is the first study that has explored these effects in detail in cross-linked PEG hydrogels using FCS; our findings question the assumption that PEG hydrogels are completely inert to protein interactions when applied as drug delivery matrices and tissue engineering scaffolds.

Multiwell stiffness assay for the study of cell responsiveness to cytotoxic drugs.

It is now well understood that the cell microenvironment, including the surrounding matrix, profoundly affects cell fate. This is especially true for solid tumors where, for example, matrix stiffness is believed to be an important factor in tumorogenesis. Our hypothesis is that since matrix stiffness affects cell fate, it may also be important in drug resistance. To test this hypothesis, we designed and built a multiwell polyacrylamide (PA) gel‐based stiffness assay, in which the gels were coated with collagen in order to facilitate cell attachment and proliferation. This PA‐based assay was used to examine the effect of stiffness on cultured cell responsiveness to cytotoxic drugs. In particular, we tested multiple cancer cell lines and their susceptibility to paclitaxel, a microtubule‐targeting agent. By assessing cell proliferation, morphology, and the IC50 of the drug, we were able to establish that the stiffness affects responsiveness to cytotoxic drugs in a cell‐dependent manner. Biotechnol. Bioeng. 2014;111: 396–403.

Protein–Hydrogel Interactions in Tissue Engineering: Mechanisms and Applications

Recent advances in our understanding of the sophistication of the cellular microenvironment and the dynamics of tissue remodeling during development, disease, and regeneration have increased our appreciation of the current challenges facing tissue engineering. As this appreciation advances, we are better equipped to approach problems in the biology and therapeutics of even more complex fields, such as stem cells and cancer. To aid in these studies, as well as the established areas of tissue engineering, including cardiovascular, musculoskeletal, and neural applications, biomaterials scientists have developed an extensive array of materials with specifically designed chemical, mechanical, and biological properties. Herein, we highlight an important topic within this area of biomaterials research, protein-hydrogel interactions. Due to inherent advantages of hydrated scaffolds for soft tissue engineering as well as specialized bioactivity of proteins and peptides, this field is well-posed to tackle major needs within emerging areas of tissue engineering. We provide an overview of the major modes of interactions between hydrogels and proteins (e.g., weak forces, covalent binding, affinity binding), examples of applications within growth factor delivery and three-dimensional scaffolds, and finally future directions within the area of hydrogel-protein interactions that will advance our ability to control the cell-biomaterial interface.

Hindered Diffusion in Polymeric Solutions Studied by Fluorescence Correlation Spectroscopy

Diffusion of molecules in the crowded and charged interior of the cell has long been of interest for understanding cellular processes. Here, we introduce a model system of hindered diffusion that includes both crowding and binding. In particular, we obtained the diffusivity of the positively charged protein, ribonuclease A (RNase), in solutions of dextrans of various charges (binding) and concentrations (crowding), as well as combinations of both, in a buffer of physiological ionic strength. Using fluorescence correlation spectroscopy, we observed that the diffusivity of RNase was unaffected by the presence of positively charged or neutral dextrans in the dilute regime but was affected by crowding at higher polymer concentrations. Conversely, protein diffusivity was significantly reduced by negatively charged dextrans, even at 0.4 μM (0.02% w/v) dextran. The diffusivity of RNase decreased with increasing concentrations of negative dextran, and the amount of bound RNase increased until it reached a plateau of ∼80% bound RNase. High salt concentrations were used to establish the electrostatic nature of the binding. Binding of RNase to the negatively charged dextrans was further confirmed by ultrafiltration.

Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds as a cell delivery vehicle: Characterization of PC12 cell response

The central nervous system (CNS) has a low intrinsic potential for regeneration following injury and disease, yet neural stem/progenitor cell (NPC) transplants show promise to provide a dynamic therapeutic in this complex tissue environment. Moreover, biomaterial scaffolds may improve the success of NPC‐based therapeutics by promoting cell viability and guiding cell response. We hypothesized that a hydrogel scaffold could provide a temporary neurogenic environment that supports cell survival during encapsulation, and degrades completely in a temporally controlled manner to allow progression of dynamic cellular processes such as neurite extension. We utilized PC12 cells as a model cell line with an inducible neuronal phenotype to define key properties of hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds that impact cell viability and differentiation following release from the degraded hydrogel. Adhesive peptide ligands (RGDS, IKVAV, or YIGSR), were required to maintain cell viability during encapsulation; as compared to YIGSR, the RGDS, and IKVAV ligands were associated with a higher percentage of PC12 cells that differentiated to the neuronal phenotype following release from the hydrogel. Moreover, among the hydrogel properties examined (e.g., ligand type, concentration), total polymer density within the hydrogel had the most prominent effect on cell viability, with densities above 15% w/v leading to decreased cell viability likely due to a higher shear modulus. Thus, by identifying key properties of degradable hydrogels that affect cell viability and differentiation following release from the hydrogel, we lay the foundation for application of this system towards future applications of the scaffold as a neural cell delivery vehicle.

Three‐dimensional matrix stiffness and adhesive ligands affect cancer cell response to toxins

There is an immediate need to develop highly predictive in vitro cell‐based assays that provide reliable information on cancer drug efficacy and toxicity. Development of biomaterial‐based three‐dimensional (3D) cell culture models as drug screening platforms has recently gained much scientific interest as 3D cultures of cancer cells have been shown to more adequately mimic the in vivo tumor conditions. Moreover, it has been recognized that the biophysical and biochemical properties of the 3D microenvironment can play key roles in regulating various cancer cell fates, including their response to chemicals. In this study, we employed alginate‐based scaffolds of varying mechanical stiffness and adhesive ligand presentation to further explore the role of 3D microenvironmental cues on glioblastoma cell response to cytotoxic compounds. Our experiments suggested the ability of both matrix stiffness and cell‐matrix adhesions to strongly influence cell responses to toxins. Cells were found to be more susceptible to the toxins when cultured in softer matrices that emulated the stiffness of brain tissue. Furthermore, the effect of matrix stiffness on differential cell responses to toxins was negated by the presence of the adhesive ligand RGD, but regained when integrin‐based cell‐matrix interactions were inhibited. This study therefore indicates that both 3D matrix stiffness and cell‐matrix adhesions are important parameters in the design of more predictive in vitro platforms for drug development and toxicity screening. Biotechnol. Bioeng. 2016;113: 443–452.

Development and characterization of polyethylene glycol–carbon nanotube hydrogel composite

Carbon nanotube (CNT)-hydrogel composites are attractive for a variety of neural tissue engineering and drug delivery applications as well as biosensor coatings, transducers and leads. Both materials contribute unique and beneficial properties to the composites. Hydrogels are an excellent mimic of the extracellular matrix due to their hydrophilicity, viscoelasticity and biocompatibility. CNTs, on the other hand, can impart electroconductivity to otherwise insulating materials, improve mechanical stability and guide neuronal cell behavior as well as elicit axon regeneration. Not surprisingly, there has been a surge in the development of various CNT-hydrogel composites including both natural and synthetic polymers. Here, we describe a CNT-polyethylene glycol (PEG) hydrogel composite where the CNTs are entrapped in the hydrogel phase during gelation. The hydrogel crosslinking reaction is based on Michael-type addition which is ideal for in situ cell and protein encapsulation. To adequately disperse the highly hydrophobic CNTs in the aqueous polymer solution, we used sonication and surfactants, where bovine serum albumin was found to be an effective and non-cytotoxic dispersant. We demonstrate that the inclusion of the CNTs impeded the hydrogel crosslinking leading to longer gelation times, higher swelling and porosity, and lower storage modulus above a threshold CNT concentration. As anticipated, composite hydrogel resistivity decreased with the incorporation of CNTs and was dependent on both CNT loading and dispersion. Importantly, unlike the PEG hydrogel alone, the PEG-CNT hydrogel composite was capable of supporting high neural cell viability where the CNTs provided sites for cell attachment.

Numerical and In Vitro Investigation of a Novel Mechanical Circulatory Support Device Installed in the Descending Aorta

Traditional implantation techniques of assist devices from the apex of left ventricle to the ascending or descending aorta are highly invasive and carry substantial complications for end-stage heart failure patients. This study has shown that the descending aorta can be a promising location to install an implantable mechanical circulatory support with minimally invasive surgery. Herein, the hemodynamic effect of an in-house prototyped pump implanted in the descending aorta was investigated numerically as well as experimentally. The objective of the experimental study is met by using the in-house simulator of the cardiovascular loop replicating congestive heart failure conditions. The objective of the numerical study was met by using the modified version of the concentrated lumped parameter model developed by the same team. The results show that the pump placement in the descending aorta can lead to an improvement in pulsatility. The pressure drop, generated at the upstream of the pump, facilitates the cardiac output as a result of after-load reduction, but at the same time, it induces a slight drop in the carotid as well as the coronary perfusion. The pressure rise, generated at the downstream of the pump, improves the blood perfusion in the renal circulation.