Browsing by Author "Segura, Tatiana"

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.

There remains a significant gap in the need for regenerative therapies for stroke compared to what is currently available. An ideal therapy would be one that stimulates the formation of new tissue with the ability to regain any function previously lost due to stroke. Therefore, methods exploiting the plasticity of the brain and modulating endogenous cellular responses to promote repair in the stroke core after ischemia have become highly attractive. However, this process of neural regeneration is complex and requires a series of controlled biological events, such as recruitment and differentiation of neuron progenitor cells (NPC’s), angiogenesis, and axonogenesis. Biomaterials are now commonly used to research tissue regeneration and cellular mechanisms, both in vitro and in vivo. We have designed a biocompatible biomaterial from macroporous annealed particles (MAP) hydrogels for injection into the stroke core five days after a photothrombotic stroke. Our hyaluronic acid-based material has been modified to dictate NPC fate in vitro through maintained stemness and the formation of neurospheres or towards Tuj1 positive NPCs, as well as enhance angiogenesis and the recruitment of endogenous NPCs after stroke. We have observed the first case of significant angiogenesis throughout the entire stroke core within only 10 days after injection, or 15 days post stroke. As well as significant increase in Tuj1+ and Nestin+ cells.

In tissues where the vasculature is either lacking or abnormal, biomaterial interventions can be designed to induce vessel formation and promote tissue repair. The porous architecture of biomaterials plays a key role in influencing cell infiltration and inducing vascularization by enabling the diffusion of nutrients and providing structural avenues for vessel ingrowth. Microporous annealed particle (MAP) scaffolds are a class of biomaterial that inherently possess a tunable, porous architecture. These materials are composed of small hydrogel particles, or microgels, that pack together to produce an interconnected, porous network. We first demonstrated that the particle fraction in MAP scaffolds serves as a bioactive cue for cell growth. To control this bioactive cue, we developed methods to form MAP scaffolds with user-defined particle fractions to reproducibly assess mechanical properties, macromolecular diffusion, as and cell responses. We then modulated the microstructure of the MAP scaffolds by changing microgel size as well as the spatial bioactivity using heterogeneous microgel populations to promote de novo assembly of endothelial progenitor-like cells into vessel-like structures. Through a combination of in silico and in vitro experimentation, we found that the microstructure (dimension of the void), integrin binding, and growth factor sequestration were all shown to guide vascular morphogenesis. We then demonstrated that the findings produced in a reductionist model of vasculogenesis translated to an in vivo effect on vessel formation in both dermal wounds and glioblastoma tumors.

Nucleic acid delivery has applications ranging from tissue engineering to biologics development and manufacturing to vaccines and infectious disease. To improve delivery and extend viable expression over time, we turn to biomaterials as a method for sustained nucleic acid release and enhanced cell culture or tissue interaction. Here, we describe how cationic polymer and lipid condensed nucleic acids can be effectively loaded into injectable granular hydrogel scaffolds by stabilizing the condensed nucleic acid into a lyophilized powder, loading the powder into a bulk hydrogel, and then fragmenting the gel into hydrogel microparticles. The resulting microgels contain non-aggregated nucleic acid particles, can be annealed into an injectable microporous scaffold, and can effectively deliver nucleic acids to cells with a sustained rate of expression. We explore how this technology can improve the production of biologics, like antibodies and viruses, to overcome limitations of current batch processes. Our scaffolds allow for continuous biologics manufacturing, with sustained production upwards of 30 days. We also explore how our platform can improve tissue regeneration in disease models like dermal wounds by delivering nucleic acid drugs, namely DNA, mRNA, and therapeutic viruses. The loaded granular scaffolds allow cells to readily repopulate the missing tissue and drugs be locally released and taken up over time. Overall, our scaffold delivery approach is a customizable platform that can be tuned for many different applications.

Our lab designs hydrogel microparticles (HMPs) that are interlinked to form microporous annealed particle (MAP) scaffolds for wound healing applications. The therapeutic effects of MAP are attributed, in part, to the void space between particles that creates inherent micro-porosity through which cells can infiltrate and migrate unhindered. Cell behavior is influenced by local geometry, and our goal is to design scaffolds that influence cells toward pro-healing behaviors. To accomplish this, we need a methodology for quantitatively characterizing the void space of MAP scaffolds in order to study the relationships between internal microarchitecture and therapeutic outcomes. The work presented here is a visually-rich dissertation that covers our approach for analyzing the void space of packed particles. We use techniques from computational geometry and graph theory to develop a robust methodology for segmenting the void space into natural pockets of open space and outputting a set of descriptors that characterize the space. Our methods are developed using simulated MAP scaffolds covering a range of particle compositions, including mixed particle sizes, stiffnesses, and shapes. Our software, called LOcal Void Analysis of MAP scaffolds (LOVAMAP), has allowed us to study many aspects of void space, including global descriptors like void volume fraction, local ‘pore’ measurements of size and shape, and additional features like ligand availability, paths, isotropy/anisotropy, and available regions for unhindered migration based on size. LOVAMAP is an enabling technology that can be used for analyzing real scaffolds or studying simulated scaffolds to inform material design. It serves as a platform for void space analysis that can easily be built upon to encompass ever-growing innovations in scaffold characterization.

When designing biomaterials for clinical applications, the performance of these platforms hinges on their interaction with the host immune system. A failure in engaging and incorporating the correct immune response would lead to foreign body response and subsequent rejection of the materials. To improve the biocompatibility of biomaterials and avoid undesired immune reactions, the key immunomodulatory cell type macrophage needs to be engaged and its phenotype modulated properly and timely. Therefore, the design parameters of biomaterials should be carefully considered in the context of macrophage modulation. Microporous annealed particle scaffolds (MAPS) are a new class of immunomodulatory granular materials generated through the interlinking of microgels. The modular nature of MAPS offers enormous tunability in not only the individual microgel design but also the homogenous or heterogenous microgel assembly into the bulk scaffold. We leveraged the plug-and-play feature of MAPS to study the effect of two design parameters, microgel crosslinking peptide (comprised of L- or D-amino acids) and spatial confinement (achieved through varying microgel size), on macrophage modulation and host responses. We uncovered that a fine balance between pro-regenerative and pro-inflammatory macrophage phenotypes in MAPS with D-amino acid-based crosslinker was an indicator for regenerative scaffolds in a subcutaneous implantation model. We also discovered that scaffolds comprised of large microgels with pore size that can accommodate ~40 µm diameter spheres induced a more balanced pro-regenerative macrophage response and better wound healing outcomes with more mature collagen regeneration and reduced inflammation level. The role of spatial confinement on macrophage response was further explored in vitro, where we demonstrated that size-dependent macrophage response to M1/M2 cytokine stimulations was tied to the change in cell morphology and motility. This work offers valuable insights into the dynamic immune response to synthetic porous scaffolds with a specific focus on macrophages, and establishes a foundation for further optimization of immunomodulatory pro-regenerative outcomes for would healing and biomaterial implants.