Tissue engineering is a combination of advanced technologies with a goal to fabricate better materials for the repair or replacement of damaged tissue. This is accomplished with the help of cells, biomaterials, and other physiological and chemical cues. In tissue engineering, human mesenchymal stem cells (hMSCs) have huge potential as they can differentiate into many cell types, reduce immune response, be isolated from adult bone marrow, and are easily available. The present study analyzes the gene expression profile of hMSCs when cultured under different conditions: two-dimensional (2D) versus three-dimensional (3D) culture systems; presence or absence of radicals, and exposure to ultra violet (UV) radiation. In our experiments, cells were cultured on 2D substrates and in 3D hydrogel systems fabricated with and without free-radicals. Total RNA was extracted from cells cultured under each condition and the differential expression of genes was analyzed using the Human U133 plus 2.0 Affymetrix gene chip. We observed that the largest difference in gene expression occurs in cells cultured in 3D as compared to 2D systems. UV radiation did not have a significant effect on gene expression but, when combined with the free-radicals generated during fabrication, significant variations in the hMSC gene expression profile were observed. Therefore, can conclude that, while all three factors (2D vs. 3D, free-radicals, and UV light) can influence the gene expression profile of hMSCs, switching from 2D to 3D cell culture system results in the largest change in gene expression.

This dissertation represents a broad exploration of polymer chemistry, anchored in the principles of the Circular Economy and sustainability. The work presented herein places a strong emphasis on utilizing monomers and polymers sourced from nature. We endeavor to enhance the utility of lignin-derived polymers for commercial applications by improving solubility and thermal properties, while also delving into the synthesis of aromatic-aliphatic polyesters from renewable resources. Lignin-derived poly(ether-amide)s are targeted initially, with the aim of overcoming their solubility limitations through thiol-ene reactions. By introducing small thiol-tagged molecules, the repeating double bonds are reduced, leading to increased chain flexibility and in turn, improved solubility and glass transition temperatures. We then explored the copolymerization of lignin-derived components with lactones for the synthesis of aromatic-aliphatic polyesters. Lactones, such as lactide and glycolide, are natural building blocks pervasive across nature. They were used in ring-opening polymerizations to produce aliphatic alcohol likers. The lactone linkers underwent condensation polymerizations with hydroxycinnamate dimers, creating sustainable, high molecular weight polyesters with desirable thermal properties. These approaches not only enhance the viability of polymers from biomass but also align with the ethos of sustainability by repurposing natural materials.

We then pivot to focus on the development of hydrogel wound dressings, as a biomimetic extracellular matrix. Bioinspired polymers were used to initiate redox hydrogel crosslinking, leading to hydrogels with improved biocompatibility compared to the gold standard approach (APS and TEMED). Finally, this was expanded to the development of in situ forming hydrogel wound dressings that sequester and release antibiotics, analgesics, and hemostatic agents. These hydrogels exhibited rapid formation, controlled release of therapeutic agents, and biocompatibility, addressing the acute need for hemostasis and infection prevention in traumatic blast wounds. In vivo studies demonstrated the safety and efficacy of these hydrogel wound dressings in reducing bacterial burden and promoting wound healing. Overall, this work presents a holistic approach to polymer science, drawing inspiration from nature and rooted in sustainability. By harnessing the potential of natural monomers and polymers, we seek to advance the field while addressing pressing challenges in materials science and healthcare.

Rising to dangerously high levels all over the world, antibiotic resistance is now a critical global problem. To inhibit or kill bacteria, most antibiotics target the growth process of bacterial cells. Therefore, most antibiotics are ineffective for slowly growing bacteria with lower cellular influx. Current therapeutic approaches most commonly utilize broad-spectrum antibiotics and/or drug cocktails to target a wide range of bacteria. Unfortunately, broad-spectrum antibiotics can target both beneficial and pathogenic bacteria. This nonspecificity imparts significant pressure on bacterial species to evolve into resistant strains. In addition, beneficial bacteria do not always fully recover, increasing susceptibility to infections and diseases. It is not practical to develop antibiotics for all species of interest. To mitigate these issues, we must engineer new antibiotics in a drastically different way to prevent running out of effective therapies. Bacteria are capable of producing defensive molecules. These secreted multi-functional molecules inhibit other closely related strains competing for the same environmental niches. We were inspired by the defensive behavior of bacteria to engineer novel antibiotics featuring tunable functionalityThis tunability allows for an adjustable bandwidth of antimicrobial activity against multiple species within a specific set of environmental conditions. Moreover, we produced antibiotics able to inhibit pathogenic bacteria without harming host cells or beneficial commensal bacteria. We were interested in antimicrobial peptides (AMPs) as antibiotics, since they target generic features common to the outer membrane of many pathogenic species. These antibiotics allow us to regulate complex microbial communities by inhibiting or killing detrimental bacteria without harming host-associated microbial communities, that have beneficial impacts on human health. Additionally, these new antibiotics help beneficial bacteria win the competition over environmental niches. Therefore, the development of resistance is significantly inhibited compared to that of conventional antibiotics. To engineer new antimicrobials, we leveraged recently developed sequence design rules, using both positive charge and hydrophobicity as necessary conditions for antimicrobial activity. These hybrid antimicrobial molecules can be used for treating resistant infections and regulating species distribution in microbial communities. We engineered tunable antimicrobial peptides with a ‘threshold’ activity profile using pH-switchable charge. To tune the antimicrobial activity of α−helical AMPs, we modified functional groups on the side chain of specific basic amino acids (lysine and arginine). By incorporating electron withdrawing and/or hydrophobic compounds on the side chains of arginine and lysine residues, the pKa at which these residues become positively charged can be lowered. Therefore, by incorporating different masking groups in different locations, we adjust the activity of the antimicrobial peptides by turning them on at specific and tunable pH values.

Prolonged field care (PFC) for treatment of battlefield and trauma injuries requires the advancement of wound management techniques in order to prevent loss of life or limb prior to hospitalization in austere combat locations where medical evacuation is delayed. The goal of this project is to design a hydrogel wound dressing capable of providing sustained release of antibiotics, analgesics, and hemostatic agents over a three-day period. Poly(ethylene glycol) (PEG) hydrogels were fabricated through crosslinking using redox initiators – ammonium persulfate (APS) and tetramethylethylene diamine (TEMED). Hydrogels were characterized through the mass swelling ratio (qm) to determine the mesh size (ξ) and thus qualitatively predict the release kinetics of the therapeutic drugs. Hydrogels with incorporated therapeutic drug were placed in known volumes of deionized water, from which aliquots were taken at set time intervals. A UV Visible Spectrophotometer determined the aliquots’ absorbance which determined the cumulative release kinetics. Ultimately, three-day sustained release of the therapeutic drugs from the PEG hydrogel was achieved through retarding the diffusion of the therapeutic drugs by incorporating acrylic acid.

Glycopolymers are synthetic analogues of natural polysaccharides that connect saccharides through a synthetic backbone rather than through glycosidic bonds. Current glycopolymerization techniques can be used to create large quantities of material with good control over the saccharide identity and chain length of the polymer, which has allowed structure-property studies of glycopolymer binding with lectins. These studies have shown that structures with longer chain length exhibit greater binding with lectins. However, these studies have not fully addressed the effects of branching or spatial orientation on lectin binding.

Branching architecture affects the biological and physical properties of polysaccharides. Similarly, branching should also affect how glycopolymers interact with their target lectins, yet few reports of branched glycopolymers have been reported. Additionally, there have been no studies on the effect of placing saccharide residues in the polymer backbone and at the branch point. To address this limitation in current synthetic techniques, polymers with branching architecture that incorporate saccharide at the branch point have been synthesized via atom transfer radical polymerization of a saccharide (either mannose or galactose) inimer and mannose monomer. Branching architecture was confirmed through GPC-PMMA, GPC-LS, and mass spectrometry. These branched glycopolymers more fully recapitulate natural branched polysaccharide structures and were found to interact more strongly than linear glycopolymers with mannose binding lectin (MBL), an immune complement protein. Most significantly, mannose at the branch point increases the polymer's interaction with MBL compared to similar structures with galactose or no saccharide content at the branch point.

In addition to polysaccharides found throughout living systems, proteins are often post-translationally modified with polysaccharide chains to create glycoproteins. We hypothesized that mimicking the 3D spatial orientation of these glycans through polymerization of glycomonomers from a protein macroinitiator can result in different binding properties of the resulting conjugate. Bovine serum albumin was modified to present multiple initiator groups which initiated the polymerization of mannose and galactose monomers via atom transfer radical polymerization. MBL interaction increases with the number and density of mannose residues attached to the protein. 3D presentation of multiple polymer chains from a protein significantly enhances lectin interaction than compared to linear glycopolymer chains with the same number of mannoses but without 3D presentation.

Structure-property studies have also been hampered by the inherently different distributions in molecular weight between different glycopolymer samples. Post-polymerization modification of pyridyl disulfide polymers with thioglycosides was demonstrated as a route towards creating glycopolymers with pendant glycosides and uniform underlying architecture and polymer chain distribution. These polymers were used to study the effect of glycopolymers on fibroblast adhesion.

We have described synthetic techniques for creating biomimetic glycopolymers that narrow the gap between synthetic structures and natural polysaccharides while still maintaining the high throughput advantage of glycopolymerizations. The structure-property studies have shown how subtle changes in polymer branching architecture and 3D spatial orientation can lead to dramatic enhancements of lectin binding, and can be applied to improved designs of glycomimetic drugs.

Chronic wounds affect many patients in the United States every year and generally coincide with other cardiovascular diseases, such as diabetes and foot ulcers. Current treatments for chronic wounds are lacking effectiveness and in the case of surgical intervention cause pain to patients. Therefore, new therapies are emerging that involve the use of hydrogels to promote natural healing by the body. Hydrogels (used as a biomaterial scaffold) have emerged as an option to treat chronic wounds, however, these scaffolds are implanted into the body and may not actually mimic the native tissue. Typically, scaffolds are completely rigid, whereas the human body is dynamic, with forces pushing and pulling constantly on tissues and cells. This thesis explored the creation of a dynamic scaffold for the repair of chronic wounds.

This work focused on creating a scaffold from modified hyaluronic acid polymer building blocks that could form after injection into the body, thus decreasing the need for surgical intervention. The scaffold contained differing ratios of cross-linkable and non-cross-linkable microspheres (or building blocks). The cross-linkable spheres form the porous network of the scaffold, while the non-cross-linkable spheres are able to move around and introduce dynamic forces into the system. In addition to the scaffold creation, a large portion of this work included optimizing the fabrication of these polymer building blocks (or microspheres). The microspheres were created using both inverse suspension and microfluidic setups. The goal was to create uniform spheres to eliminate the possible effect differing geometries could have on the scaffolds. The observable difference in the scaffolds was thus the amount of cross-linked spheres and non-cross-linked spheres.

After fabrication of the microspheres, a scaffold was created by mixing the microspheres together with a cross-linker and characterized through Rheometry, and microscopy. After analysis, the time and minimum amount of cross-linkable spheres required for gelation to occur in the scaffolds was determined to be 1 hour and 30 minutes and 50% respectively. Additionally, it was observed that after decreasing the amount of cross-linkable spheres below 80%, the overall scaffold strength and modulus significantly decreased. Lastly, it was observed that the void space or free space in the scaffolds did not significantly change between any of the scaffold conditions. Following physical characterization, the scaffolds were cultured with human dermal fibroblasts to determine how the dynamic scaffold would interact with the cells. For all the dynamic scaffolds, as well as the control scaffold (a completely cross-linked porous microsphere scaffold) there was an increase of proliferation and spreading over the 7-day trial. Although there was improvement in all the conditions, there was no statistical difference seen between the conditions. Thus, it was concluded that all these scaffolds are biocompatible and can improve cell growth; however, at the moment it cannot be stated that a dynamic scaffold has improvement over the traditionally cross-linked scaffold.

The field of cell and tissue engineering is far from being systematic. Historically, the field follows a guess-and-test methodology; new materials are produced and tested in search of the “right” combination of material properties, chemical/growth factor concentrations, reactor conditions, etc. While developmental biologists have extensively studied signaling factors, gene expression and other components governing early tissue development, researchers still do not have a full picture of how these signaling cascades are initiated or how spatial and temporal tissue inhomogeneities initially form. To address this issue, new materials must be developed that can mimic the intricacies of native tissue in order to correctly study their behavior. Hydrogels incorporating controlled photodegradation are a novel class of polymeric biomaterials that our group at UCLA has developed. The outstanding benefit of these materials is that their physical and chemical properties can be altered on-demand, in real-time without the presence of toxic compounds, allowing cells to be present during modification. In addition, the degradation, and thus the mechanical properties, is a strict function of the exposure of light (exposure time, wavelength, and intensity) such that the precise spatial and temporal control of the degree of degradation far surpasses that of hydrolytic and enzymatic degradation mechanisms. In this dissertation, I develop new photodegradable materials, find solutions to better characterize their behavior, and expand the techniques necessary for their successful use in cell biology. First, by developing a series of mass-action and kinetic mathematical models, I examine the physical properties of photodegradable gels formed by end-linking gelation. I pay special attention to how diffusion of photoabsorbing byproducts affect degradation. These models are further enhanced by examining the specific microstructure and micro-heterogeniety of the gels formed. Second, I expand the photodegradable materials library. In order to better mimic three-dimensional cellular environments, I successfully synthesize photodegradable protein-based gels and showcase their applicability towards three-dimensional cell culture. I specifically fabricate photodegradable gelatin gels, however, the techniques I develop here more generally aid in the conjugation of hydrophobic moieties to protein materials. Next, I synthesize and fabricate photodegradable polyacrylamide gels. I utilize the flexibility of the polyacrylamide gel system by exploring cell response to both changes in cell binding domain and dynamic softening of the underlying matrix. Finally, I develop the application of maskless photolithography for photodegradable hydrogels. Using this technique, I rapidly pattern grayscale stiffness patterns into photodegradable hydrogels in a highly controlled fashion with sub-micron resolutions. Cell response to complex patterns of grayscale stiffness are tested. Through the developments made in this dissertation, I expand our ability to test cell behavior in spatially and temporally heterogeneous environments.

In embryogenesis and morphogenesis, cell clusters or sheets organize into units of increasing complexity from multi-cell layers, to tissues, organs and eventually whole organisms. Dynamic changes in shape are an essential part of morphogenesis and even the most complex biological structures are formed from fairly simple units through numerous shape transformation steps. Although nature has found ways to elegantly exploit shape transformation in plants and animals, the field is much newer to polymer scientists and engineers. Furthermore, tissue engineers are only starting to explore shape transformation for cell and tissue culture. Current shape changing scaffolds are not permissive of self-folding actuation at arbitrary time points nor are they permissive of step-wise folding, similar to what is observed during morphogenesis and development. The subject of this manuscript is on shape-changing photodegradable hydrogels for cell and tissue culture. We first discuss the physical basis for how light is used to pattern differences in swelling throughout a hydrogel film. The differences in swelling are balanced by the hydrogel’s network elasticity, resulting in folding of free-floating hydrogels. Beyond demonstrating shape-change as a proof of concept, we systematically review the effect of material, chemical, and physical parameters on curvature. Next, we discuss the use of planar sheet geometries and spatiotemporal light patterning to actuate hydrogel folding with different mean and Gaussian curvatures that are relevant for human biology. Finally, we demonstrate that the hydrogels are capable of supporting 2D seeded cell culture and 3D encapsulation. Mammalian cells on the hydrogel scaffolds are shape changed at arbitrary time points and remain viable. We thus demonstrate photodegradable hydrogel shape change as a viable concept for dynamic, 4D cell culture.

Polysaccharides are polymeric chains comprised of saccharides connected through glycosidic bonds that have important biological functions, such as energy storage, structure, and cell signaling. Although polysaccharides are ubiquitous in nature, there are limited tools for examining their roles due to the complexity of polysaccharide synthesis. In order to synthesize well-defined polysaccharides, selective protection/deprotection techniques are employed that require tedious purification at each intermediate. In order to facilitate the creation of high molecular weight saccharide-bearing polymers, glycopolymers can be created as an alternative. Glycopolymers emulate the interactions observed in natural polysaccharides through the use of synthetic analogues.

Glycopolymers are synthetic polymers that present saccharide side groups. Although glycopolymers have been produced with control over saccharide identity and molecular weight, only limited investigations have been conducted to incorporate cationic charge or control branched architecture. Currently, cationic charge is incorporated through the copolymerization of cationic monomers with glycomonomers, but the inclusion of cationic monomers has led to increased cytotoxicity. There have been no examples of glycopolymers utilizing the inherent charge available on amino-sugars, such as glucosamine. In addition, branched glycopolymers have previously been studied using atom-transfer radical polymerization, but the polymerization technique is incompatible with amine-containing monomers and requires removal of the copper catalyst. Reversible addition-fragmentation chain transfer (RAFT) polymerization is an alternative technique for glycopolymer synthesis, but branching has only been introduced using crosslinkers that result in gelation at high degrees of incorporation. To address these limitations, new cationic glycomonomers and RAFT branching units have been synthesized.

Using these new tools, in addition to those currently available for glycopolymer synthesis, a number of biomedical applications were investigated: bacterial attachment on glycopolymer-modified surfaces, lectin interaction with 3D constructs that present saccharides at different densities, antibacterial activity of a cationic glycopolymer, and potential usage of a cationic glycopolymer in gene transfection.

Using a set of four glycomonomers synthesized from glucose, galactose, mannose, and N acetyl glucosamine, glycopolymer of various saccharide identities were polymerized via RAFT polymerization and conjugated to gold surfaces to investigate the attachment of Shewanella oneidensis and Vibrio cholerae. Polymeric mannose was seen to encourage significantly more attachment than monomeric mannose, polymeric galactose, and polymeric N acetyl glucosamine.

Using a RAFT branching unit, amphiphilic glycopolymers were polymerized and self-assembled into nanoparticles with control over the surface saccharide density without affecting size and morphology. The nanoparticles with increased branching bound more lectin compared to nanoparticles without branching and glycopolymers in solution.

Using a cationic glycomonomer, a chitosan-mimic was polymerized and investigated for similarities in bioactivity. In antibacterial studies, the cationic glycopolymer closely mimicked the ability of chitosan to inhibit bacterial growth above a threshold molecular weight. Unlike chitosan, the glycopolymer was also soluble in neutral and basic buffer and maintained its ability to inhibit bacterial growth. As a transfection agent, the glycopolymer was able to induce gene expression with less cytotoxicity than poly(ethylenimine), the standard synthetic transfection agent, but with more cytotoxicity than chitosan. With optimization, the chitosan-mimic can be an efficient transfection agent with low cytotoxicity.

We have described the addition of new synthetic tools for creating cationic glycopolymers and hyperbranched glycopolymers. The applications presented demonstrate the importance of control over saccharide identity, branching, and charge in glycomimetic systems and can be applied to the design of new therapeutics and devices.

Cationic polymers are of broad interest in the biotechnology sector due to their potential use in drug conjugation, tissue engineering, gene delivery, water purification and chelation, and stimuli-responsive materials. Of particular interest in designing cationic polymers is control over their pKa—allowing for the presentation of a positive charge at specific pH, which is dependent on molecular weight, connectivity, and pendant group identity of the polymer. In this dissertation, we designed and synthesized a series of smoking-inspired monomers and polymers based on pyrrolidine, piperidine, and tocopherol—utilizing copolymerization and other polymer chemistry strategies to vary their molecular weight, dispersity, hydrophilicity/hydrophobicity, charge density, and pKa. These polymers are designed to mimic certain chemical structures present in tobacco alkaloids or other smoking-related compounds that have implications in human health. In this work, we explore the utility of amine-based polymers within and beyond the scope of innate immunity—playing important roles in solving bioengineering and tissue engineering problems. Exposure to certain chemical factors present in tobacco and smoking-related compounds leads to the elevated risk and greater symptom severity of several autoimmune conditions. While this link has remained mechanistically unclear, the presence of protein/DNA immune complexes in systemic lupus erythematosus (SLE) has been designated as an important marker of disease severity, seen more prevalently in smokers vs. never-smokers. The smoking-inspired polymer library developed in this study is used to mimic the features of these immune complexes. Investigation of these polymeric materials complexed with negatively-charged biological macromolecules shows notable differences in complex formation, biophysical structure, and subsequent innate immune activity. These results provide further evidence of the role of biophysical complex structure in innate immune activity, and the role that tobacco’s chemical structures may play in exacerbating disease. Additional work explores the interaction of DNA with cigarette smoke in vitro—demonstrating the formation of visible clew-like precipitates upon DNA exposure to cigarettes. These pellets, which resemble the precipitates formed by the condensation of oppositely-charged biomacromolecules, demonstrate a level of biophysical ordering and immune activity seen in immunomodulatory DNA complexes.