Browsing by Subject "Hydrogel"

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior.

In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration.

Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines.

Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.

It has been understood for some time that cells are profoundly influenced by their environment. Recently, researchers have made great strides in engineering cell culture platforms that are both physiologically mimetic and reductionist, leading to more biologically relevant cell responses observed within analytically tractable experiments. Moving from stiff tissue culture plastic or glass into hydrogel-based cell culture keeps cells in contact with materials that are stiffness-matched to native tissues, and can reduce or delay de-differentiation into undesirable phenotypes. Incorporating biomolecules like specific adhesion ligands or growth factors into cell culture hydrogels can help drive specific biological outputs, and when coupled with patterning techniques can yield intentional, spatially defined heterogeneity within a cultured cell population. However, biological events or disease states are often are driven by biochemical dynamics, with the time course over which a signal is presented influencing whether cells respond physiologically or pathologically. Unfortunately, dynamic presentations of biomolecules are challenging to replicate within hydrogels due to a lack of ligation mechanisms suitable for dynamically linking relevant biomolecules into the hydrogel matrix in the presence of cells.

In this work, novel protein-based ligation domains were engineered into new strategies for the time-varying presentation of recombinant biomolecules within cell culture hydrogels. SpyCatcher, a protein domain which forms a spontaneous covalent bond with a complementary peptide dubbed “SpyTag”, was used to form a site-specific linkage between a recombinant protein and a synthetic hydrogel. The ligation reaction was shown to proceed under mild conditions appropriate for cell culture, and by adding the cell-adhesive ligand RGDS to the SpyCatcher-tagged protein (forming RGDS-SC), cell spreading within a 3D hydrogel could be switched on by simply adding RGDS-SC topically to cell-laden hydrogels. SnoopCatcher, the chemically orthogonal cousin to SpyCatcher that binds the peptide “SnoopTag”, was then appended with the vascular endothelial growth factor-mimetic peptide QK (KLTWQELYQLKYKGI) to form QK-SnpC, and used in tandem with RGDS-SC to simultaneously control endothelial cell adhesion and mitotic stimulation on synthetic hydrogels containing both Tag peptides.

The Catcher/Tag systems are advantageous due to their specificity and stability. However, their ligations are irreversible because they form covalent bonds. Incorporating the photocleavable fluorophore PhoCl into the backbone of RGDS-SC (forming PhoCl-SC) allowed for the reversible incorporation of a recombinant protein into synthetic hydrogels. SpyCatcher mediated the spontaneous ligation to SpyTag sites within the gel, and by applying 400 nm light, PhoCl was cleaved, thereby removing the N-terminal RGDS tag from the gel. This reversion was limited to one cycle, and so would not be appropriate for presenting a sequence of several biochemical signals, as would be seen by cells near an area of wound healing for instance. To develop a truly reversible conjugation mechanism, the optogenetic protein LOV2 was engineered into a blue light-mediated non-covalent ligation strategy. The LOVTRAP system, consisting of LOV2 and its binding partner Zdk, was shown to allow synthetic gels containing LOV2 to capture Zdk-tagged proteins in the dark, and then release them upon blue light exposure. Because LOV2 will reset to its dark state via thermal relaxation, this capture and release cycle could be repeated at least 3 times, indicating the reversible association of LOVTRAP was functional in a biomaterial setting.

Protein-based ligation domains are simple to use, as they can be added to recombinant proteins genetically, and are inherently site-specific, alleviating worries that protein activity could be compromised upon conjugation to the gel. Moreover, the Catcher systems and LOVTRAP all bind to their binding partners spontaneously under cell culture conditions, reducing the chance that sensitive cell types would be perturbed by their use. These strategies greatly expand the tool kit for dynamically presenting biomolecules to cells via hydrogel immobilization.

Wound healing is a vastly complicated process. While this can be said about many biological functions in the body, wounds present a particularly difficult problem due to their inherent irregularity or uniqueness. Because different wounds behave and heal differently, or not at all, different therapies must be developed to treat them effectively. The research presented here details several approaches to progress not only the entire field of wound healing research, but also focuses on hydrogel technology improvements. Using titanium 3D printing, cap-able splints were constructed to not only ease the surgical process but also enable efficient daily wound access for treatment administration or wound tracking over time without the need to completely undress and redress the wound. The titanium splints did prove effective for daily monitoring but did still require some surgical prowess. To remove the need for surgical skills, an adhesive wound splint was developed by incorporating ethoxylated polyethyleneimine (EO-PEI) into the traditional polydimethylsiloxane (PDMS) polymer recipe resulting in adhesive PDMS (aPDMS). The aPDMS splints drastically reduced surgery time per animal without compromising wound splinting performance. Traditional bulk hydrogels have been used in wound healing research but have yet to be clinically implemented in a widespread manner due in part to their resistance to cellular infiltration or integration with the host. Using hyaluronidase (HAase) on a hyaluronic acid (HA) based hydrogels to partially degrade the surface of bulk gels yielded a looser nano-scale mesh size that enhanced cellular infiltration into the gel and granted better access to nanoparticle therapy loaded within. Finally, a biologically active viscous salve loaded with heavy chains (HC) of the serum protein Inter-α Inhibitor (IαI) was designed to leverage HC’s ability to mitigate the inflammatory response such that normal wound healing regeneration could ensue.

Appropriately modulating inflammation after traumatic brain injury (TBI) may prevent disabilities in the millions that suffer TBI every year. Important mediators of inflammation include macrophages and microglia and these cell types can possess a range of phenotypes. An anti-inflammatory, “M2-like” macrophage phenotype after TBI is associated with neurogenesis, axonal regeneration, and improved white matter integrity. To boost these subpopulations, a promising approach is the enrichment of two cytokines: Fractalkine (FKN, CX3CL1) or Interleukin-4 (IL-4). FKN is a chemokine and thus recruits non-classical monocytes which are precursors to M2-like macrophages. IL-4 polarizes and proliferates M2-like macrophages. However, delivering recombinant or purified cytokines is not ideal due to their short half-lives, suboptimal efficacy, immunogenic potential, batch variabilities, and cost. Here we explore two strategies to enrich endogenous FKN or IL-4, obviating the need for delivery of exogenous proteins.

In the first study, we synthesize a biomaterial to elevate endogenous FKN at an injury site. Modified FKN-binding-aptamers are integrated with poly(ethylene glycol) diacrylate to form aptamer-functionalized hydrogels (“aptagels”) that dramatically enrich and passively release FKN in vitro for at least one week. Implantation in a mouse model of excisional skin injury demonstrates that aptagels enrich endogenous FKN and stimulate local increases in non-classical monocytes and M2-like macrophages.

In our second approach, we augment mesenchymal stem/stromal cells (MSCs), to transiently express IL-4. As MSCs do not endogenously synthesize IL-4, we transfect them with synthetic IL-4 mRNA. We suggest that mRNA transfection is a better strategy than DNA transfection, viral transduction, and recombinant IL-4 delivery for TBI. Our studies first characterize the IL-4 expression. Then, in a TBI model of closed head injury, we observe that IL-4 expressing MSCs successfully induce a robust M2-like macrophage phenotype and promote anti-inflammatory gene expression. Curiously, this does not translate to improvements in function, histology, or white matter integrity.

The results demonstrate that orchestrators of inflammation can be manipulated without delivery of foreign proteins. Both FKN-aptamer functionalized biomaterials and IL-4 expressing MSCs may be promising approaches to boost anti-inflammatory subpopulations at sites of injury. However, our studies also begin to question whether M2-like macrophages alone orchestrate the neurogenesis, axonal regeneration, and improved white matter integrity that has previously been observed.

Finally, both strategies could have important immunomodulatory roles outside of TBI. Aptagels are readily synthesized, highly customizable and could combine different aptamers to treat complex diseases in which regulation or enrichment of multiple proteins may be therapeutic. IL-4 expressing MSCs could assist tissue regeneration in cavitary diseases or improve biomaterial integration into tissues.

This thesis explores the application of nanofibrous and microfibrous materials in the fields of cartilage repair and water electrolysis.

Articular cartilage lesions have a limited intrinsic ability to heal and are associated with joint pain and disability. The current treatment options suffer from high failure rates, prolonged rehabilitation times, and can be very costly. Therefore, an ideal solution is a low cost, mechanically strong, biocompatible replacement material with long lifetime.

To develop a cartilage replacement material, I first developed a two-step method to 3D print double network hydrogels at room temperature with a low-cost ($300) 3D printer. A first network precursor solution was made 3D printable via extrusion from a nozzle by adding a layered silicate to make it shear-thinning. After printing and UV curing, objects were soaked in a second network precursor solution and UV-cured again to create interpenetrating networks of poly(2-acrylamido-2-methylpropanesulfonate) and polyacrylamide. By varying the ratio of polyacrylamide to cross-linker, the trade-off between stiffness and maximum elongation of the gel can be tuned to yield a compression strength and elastic modulus of 61.9 and 0.44 MPa, respectively, values that are greater than those reported for bovine cartilage. The maximum compressive (93.5 MPa) and tensile (1.4 MPa) strengths of the gel are twice that of previous 3D printed gels, and the gel does not deform after it is soaked in water. By 3D printing a synthetic meniscus from an X-ray computed tomography image of an anatomical model, I demonstrate the potential to customize hydrogel implants based on 3D images of a patient’s anatomy.

On the basis of the previous work, I developed the first hydrogel with the strength and modulus of cartilage in both tension and compression, and the first to exhibit cartilage-equivalent tensile fatigue at 100,000 cycles. These properties were achieved by infiltrating a bacterial cellulose nanofiber network with a PVA-PAMPS double network hydrogel. The bacterial cellulose provided tensile strength in a manner analogous to collagen in cartilage, while the PAMPS provided a fixed negative charge and osmotic restoring force similar to the role of aggrecan in cartilage. The hydrogel has the same aggregate modulus and permeability as cartilage, resulting in the same time-dependent deformation under confined compression. The hydrogel is not cytotoxic, has a coefficient of friction 45% lower than cartilage, and is 4.4 times more wear-resistant than a polyvinyl alcohol hydrogel. The properties of this hydrogel make it an excellent candidate material for replacement of damaged cartilage.

In the field of water electrolysis, I studied the effect of fiber dimensions to their performance in water electrolysis. Water electrolysis is a good way to convert excess renewable energy to hydrogen. The generation of renewable electricity is variable, leading to periodic oversupply. Excess power can be converted to hydrogen via water electrolysis, but the conversion cost is currently too high. One way to decrease the cost of electrolysis is to increase the maximum productivity of electrolyzers. I investigated how nano- and microstructured porous electrodes could improve the productivity of hydrogen generation in a zero-gap, flow-through alkaline water electrolyzer. Three nickel electrodes—foam, microfiber felt, and nanowire felt—were studied to examine the tradeoff between surface area and pore structure on the performance of alkaline electrolyzers. Although the nanowire felt with the highest surface area initially provided the highest performance, this performance quickly decreased as gas bubbles were trapped within the electrode. The open structure of the foam facilitated bubble removal, but its small surface area limited its maximum performance. The microfiber felt exhibited the best performance because it balanced high surface area with the ability to remove bubbles. The microfiber felt maintained a maximum current density of 25,000 mA cm-2 over 100 hrs without degradation, which corresponds to a hydrogen production rate 12.5- and 50-times greater than conventional proton-exchange membrane and alkaline electrolyzers, respectively.

Soft biomaterials are used in a variety of applications such as scaffolds for cell growth and coatings for implants or transplants. We aim to create a protein hydrogel that will self-assemble upon the mixing of two different protein constructs. This is accomplished using Streptavidin, a protein that tetramerizes, and SpyTag-SpyCatcher, a protein-peptide that spontaneously forms covalent bonds, as the crosslinking mechanisms. Further, using protein building blocks whose viscoelastic properties are known from single-molecule force spectroscopy (SMFS), we aim to create a hydrogel whose physical properties are tailorable by altering the building blocks incorporated in the constructs. This thesis focuses on using an atomic force microscope for force spectroscopy and imaging to analyze the formation of networks upon mixing various protein constructs. We find that small scale networks form using both Streptavidin and SpyTag-SpyCatcher as crosslinkers and that SpyCatcher can be used to detect molecular crosslinking and polyprotein polarization through SMFS.

There are approximately 900,000 people in the US suffering from damage to the articular cartilage, with the knee being most commonly affected. Articular cartilage lacks a vasculature and has a limited ability to heal. A variety of surgical treatments have been developed to repair cartilage lesions. Current strategies for cartilage repair include microfracture, autologous chondrocyte implantation (ACI) and osteochondral allograft transfer (OAT). These strategies suffer from high failure rates (25-50% at 10 years), long rehabilitation times (more than 12 months) and decreasing efficacy in patients older than 40-50 years. Focal joint resurfacing with traditional orthopedic materials is being explored as an alternative strategy, but due to their high stiffness and coefficient of friction relative to cartilage, these implants may ultimately contribute to joint degeneration through abnormal stress and wear. A focal joint resurfacing method that is widely available, allows immediate weight bearing, has short recovery times and has low long-term failure rates remains an unmet need.This thesis explores a strategy to address this need. There are two major criteria within this strategy: 1) develop a material that mimics the properties of cartilage and 2) attach this material to an orthopedic base to enable integration with bone. I developed the first hydrogel to achieve the strength and modulus of cartilage in both tension and compression properties. This hydrogel also exhibits cartilage-equivalent tensile fatigue at 100,000 cycles. The hydrogel was created by infiltrating a PVA-PAMPS double-network hydrogel into a bacterial cellulose (BC) nanofiber network. The BC fibers provide tensile strength in a manner analogous to collagen in cartilage. The PAMPS provides a fixed negative charge and osmotic restoring force similar to the role of aggrecan in cartilage. Subsequently, I further improved and developed the hydrogel to reach a strength that exceeds that of cartilage. The high strength was achieved through reinforcement of crystallized PVA with BC. Experimental results show that reinforcement of annealed PVA with BC leads to a 3.2-fold improvement in the tensile strength (from 15.6 to 50.5 MPa) and a 1.7-fold increase in the compressive strength (from 56.7 to 95.4 MPa). The BC-reinforced PVA was also 3 times more wear resistant than cartilage over 1 million cycles and exhibited the same coefficient of friction. These properties make the BC-reinforced BC hydrogel an excellent candidate material for replacement of damaged cartilage. Current strategies for adhering hydrogel to a surface are 10 times weaker than the shear strength with which cartilage is attached to bone. The osteochondral junction is characterized by mineralized collagen nanofibers anchoring cartilage to bone. I sought to mimic this strategy by bonding freeze-dried BC to porous titanium with a hydroxyapatite-forming cement. The cement penetrates about 10 microns into the bacterial cellulose, forming a nanofiber-reinforced zone of adhesion. The PVA-PAMPS hydrogel is then infiltrated into the bonded bacterial cellulose. This strategy achieved a shear strength of attachment three times greater than the state of the art. I soon proposed an important enhancement of the attaching strategy by introducing shape memory alloy ring to change the direction of shear load bearing. It is the first method for attaching a hydrogel to metal with the same shear strength as the cartilage-bone interface. The average shear strength of the junction between 1.2-mm-thick hydrogel and metal made in this manner exceeded the shear strength of porcine cartilage-bone interface. The shear strength of attachment increased with the number of bacterial cellulose layers and with the addition of cement between the bacterial cellulose layers. Such improved strategies for attaching hydrogels to a metal surface with sufficient strength to allow for weight-bearing can enable the creation of hydrogel-capped titanium implants for cartilage repair.

Liver tissue engineering has made tremendous progress over the last decades, but continues to be limited by dedifferentiation of hepatocytes and insufficient vascularization of engineered constructs.1 Functional hepatocytes are the parenchymal liver cells and they are responsible for many of the over 500 essential liver functions. Thus, they are necessary for advancement toward effective liver disease treatments, predictive models for drug screening, and whole-organ tissue engineering.2

Liver-specific functions of hepatocytes are known to be regulated by complex microenvironmental cues that are not fully understood and are difficult to recapitulate in vitro. These signals are known to include homotypic interactions with other hepatocytes, heterotypic interactions with non-parenchymal cells, biochemical cues from surrounding extracellular matrix, and signals and sustenance from the underlying vasculature.3 The objective of this work was to investigate material properties and culture conditions that are conducive to the long-term culture of functional primary hepatocytes in order to design a platform capable of supporting them. We found that coculturing hepatocytes in adhesive and degradable hydrogels with capillary forming endothelial cells (EC) and supporting pericytes formed vascularized liver tissue-like constructs. Over time, we found that hepatocytes and vascular cells in these constructs formed close synergistic associations. While hepatocytes enhanced vascularization, microvascular networks supported at least 3 major hepatocyte functions for at least 2 months.

Then we translated our platform into a bioartificial liver for acute liver failure therapy. Bioartificial livers, are extracorporeal life-saving treatments similar to dialysis for patients with acute liver failure who are awaiting a transplant. These devices often to employ hepatocytes in cartridges filled with semi-porous hollow fibers to perfuse patient blood and temporarily off-load the patient’s liver functions, reducing blood toxicity and preserving the function of other organs in the body while the liver repairs itself. However, they are limited in that the hepatocytes used are either short-lived hepatocytes or hepatocyte derivatives that have lost essential functions during their transformation. They are also enormously large, costly, challenging to maintain, and difficult to use. Since our platform is prevascularized and capable of supporting hepatocyte function, we examined its potential to alleviate some of these issues.

Not only were ECs and pericytes able to form robust vascular networks in the context of a perfused hollow fiber-based cartridge, but they were able to anastomose to the hollow fibers and enhance mass transport of nutrients into the surrounding hydrogel and hepatocytes. Thus, we developed a module that effectively bridges macro-to-micro scale mass transport using hollow fibers as conduits for efficient, artery scale fluid transport and cell-assembled microvasculature to perfuse the engineered tissue and facilitate exchange of nutrients and metabolic waste with functional cells, interstitial fluids, and the bulk fluid from the hollow fiber. Upon the addition of hepatocytes in the bioreactor platform, we observed maintenance of liver synthetic and metabolic functions for at least 1 month, the length of time it takes a severely damaged liver to regenerate without further complications. The findings in this work serve as a proof-of-concept for future studies that further optimize the bioreactor further and examine its potential toward viable liver disease treatments.