Gene Delivery From Granular Scaffolds for Tunable Biologics Manufacturing.

The understanding of the molecular basis for disease has generated a myriad of therapeutic biologics, including therapeutic proteins, antibodies, and viruses. However, the promise that biologics can resolve currently incurable diseases hinges in their manufacturability. These therapeutics require that their genetic material be introduced to mammalian cells such that the cell machinery can manufacture the biological components. These are then purified, validated, and packaged. Most manufacturing uses batch processes that collect the biologic a few days following genetic modification, due to toxicity or difficulty in separating product from cells in a continuous operation, limiting the amount of biologic that can be produced and resulting in yearlong backlogs. Here, a scaffold-based approach for continuous biologic manufacturing is presented, with sustained production of active antibodies and viruses for 30 days. The use of scaffold-based biologic production enabled perfusion-based bioreactors to be used, which can be incorporated into a fully continuous process.

Optimizing neurotransmitter pathway detection by IR-MALDESI-MSI in mouse brain.

Mass spectrometry imaging (MSI) platforms such as infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) are advantageous for a variety of applications, including elucidating the localization of neurotransmitters (NTs) and related molecules with respect to ion abundance across a sample without the need for derivatization or organic matrix application. While IR-MALDESI-MSI conventionally uses a thin exogenous ice matrix to improve signal abundance, it has been previously determined that sucrose embedding without the ice matrix improves detection of lipid species in striatal, coronal mouse brain sections. This work considers components of this workflow to determine the optimal sample preparation and matrix to enhance the detection of NTs and their related metabolites in coronal sections from the striatal region of the mouse brain. The discoveries herein will enable more comprehensive follow-on studies for the investigation of NTs to enrich biological pathways and interpretation related to neurodegenerative diseases and ischemic stroke.

Enhanced Detection of Charged N-Glycans in the Brain by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometric Imaging.

N-linked glycosylation represents a structurally diverse, complex, co- and posttranslational protein modification that bridges metabolism and cellular signaling. Consequently, aberrant protein glycosylation is a hallmark of most pathological scenarios. Due to their complex nature and non-template-driven synthesis, the analysis of glycans is faced with several challenges, underlining the need for new and improved analytical technologies. Spatial profiling of N-glycans through direct imaging on tissue sections reveals the regio-specific and/or disease pathology correlating tissue N-glycans that serve as a disease glycoprint for diagnosis. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a soft hybrid ionization technique that has been used for diverse mass spectrometry imaging (MSI) applications. Here, we report the first spatial analysis of the brain N-linked glycans by IR-MALDESI MSI, leading to a significant increase in the detection of the brain N-sialoglycans. A formalin-fixed paraffin-embedded mouse brain tissue was analyzed in negative ionization mode after tissue washing, antigen retrieval, and pneumatic application of PNGase F for enzymatic digestion of N-linked glycans. We report a comparative analysis of section thickness on the N-glycan detection using IR-MALDESI. One hundred thirty-six unique N-linked glycans were confidently identified in the brain tissue (with an additional 132 unique N-glycans, not reported in GlyConnect), where more than 50% contained sialic acid residues, which is approximately 3-fold higher than the previous reports. This work demonstrates the first application of IR-MALDESI in N-linked glycan imaging of the brain tissue, leading to a 2.5-fold increase in the in situ total brain N-glycan detection compared to the current gold standard of positive-mode matrix-assisted laser desorption/ionization mass spectrometry imaging. This is also the first report of the application of the MSI toward the identification of sulfoglycans in the rodent brain. Overall, IR-MALDESI-MSI presents a sensitive glycan detection platform to identify tissue-specific and/or disease-specific glycosignature in the brain while preserving the sialoglycans without any chemical derivatization.

Void Volume Fraction of Granular Scaffolds.

Void volume fraction (VVF) is a global measurement frequently used to characterize the void space of granular scaffolds, yet there is no gold standard by which to measure VVF in practice. To study the relationship  between VVF and particles of varying size, form, and composition, a library of 3D simulated scaffolds is used. Results reveal that relative to particle count, VVF is a less predictable metric across replicate scaffolds. Simulated scaffolds are used to explores the relationship between microscope magnification and VVF, and recommendations are offered for optimizing the accuracy of approximating VVF using 2D microscope images. Lastly, VVF of hydrogel granular scaffolds is measured while varying four input parameters: image quality, magnification, analysis software, and intensity threshold. Results show that VVF is highly sensitive to these parameters. Overall, random packing produces variation in VVF among granular scaffolds comprising the same particle populations. Furthermore, while VVF is used to compare the porosity of granular materials within a study, VVF is a less reliable metric across studies that use different input parameters. VVF, a global measurement, cannot describe the dimensions of porosity within granular scaffolds, and the work supports the notion that more descriptors are necessary to sufficiently characterize void space.

Activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing.

Microporous annealed particle (MAP) scaffolds are flowable, in situ crosslinked, microporous scaffolds composed of microgel building blocks and were previously shown to accelerate wound healing. To promote more extensive tissue ingrowth before scaffold degradation, we aimed to slow MAP degradation by switching the chirality of the crosslinking peptides from L- to D-amino acids. Unexpectedly, despite showing the predicted slower enzymatic degradation in vitro, D-peptide crosslinked MAP hydrogel (D-MAP) hastened material degradation in vivo and imparted significant tissue regeneration to healed cutaneous wounds, including increased tensile strength and hair neogenesis. MAP scaffolds recruit IL-33 type 2 myeloid cells, which is amplified in the presence of D-peptides. Remarkably, D-MAP elicited significant antigen-specific immunity against the D-chiral peptides, and an intact adaptive immune system was required for the hydrogel-induced skin regeneration. These findings demonstrate that the generation of an adaptive immune response from a biomaterial is sufficient to induce cutaneous regenerative healing despite faster scaffold degradation.

Enhanced In Vivo Delivery of Stem Cells using Microporous Annealed Particle Scaffolds.

Delivery to the proper tissue compartment is a major obstacle hampering the potential of cellular therapeutics for medical conditions. Delivery of cells within biomaterials may improve localization, but traditional and newer void-forming hydrogels must be made in advance with cells being added into the scaffold during the manufacturing process. Injectable, in situ cross-linking microporous scaffolds are recently developed that demonstrate a remarkable ability to provide a matrix for cellular proliferation and growth in vitro in three dimensions. The ability of these scaffolds to deliver cells in vivo is currently unknown. Herein, it is shown that mesenchymal stem cells (MSCs) can be co-injected locally with microparticle scaffolds assembled in situ immediately following injection. MSC delivery within a microporous scaffold enhances MSC retention subcutaneously when compared to cell delivery alone or delivery within traditional in situ cross-linked nanoporous hydrogels. After two weeks, endothelial cells forming blood vessels are recruited to the scaffold and cells retaining the MSC marker CD29 remain viable within the scaffold. These findings highlight the utility of this approach in achieving localized delivery of stem cells through an injectable porous matrix while limiting obstacles of introducing cells within the scaffold manufacturing process.

Dual-function injectable angiogenic biomaterial for the repair of brain tissue following stroke.

Stroke is the primary cause of disability due to the brain's limited ability to regenerate damaged tissue. After stroke, an increased inflammatory and immune response coupled with severely limited angiogenesis and neuronal growth results in a stroke cavity devoid of normal brain tissue. In the adult, therapeutic angiogenic materials have been used to repair ischaemic tissues through the formation of vascular networks. However, whether a therapeutic angiogenic material can regenerate brain tissue and promote neural repair is poorly understood. Here we show that the delivery of an engineered immune-modulating angiogenic biomaterial directly to the stroke cavity promotes tissue formation de novo, and results in axonal networks along thee generated blood vessels. This regenerated tissue produces functional recovery through the established axonal networks. Thus, this biomaterials approach generates a vascularized network of regenerated functional neuronal connections within previously dead tissue and lays the groundwork for the use of angiogenic materials to repair other neurologically diseased tissues.

Pathways Governing Polyethylenimine Polyplex Transfection in Microporous Annealed Particle Scaffolds.

Gene delivery using injectable hydrogels can serve as a potential method for regulated tissue regeneration in wound healing. Our microporous annealed particle (MAP) hydrogel has been shown to promote cellular infiltration in both skin and brain wounds, while reducing inflammation. Although the scaffold itself can promote healing, it is likely that other signals will be required to promote healing of hard-to-treat wounds. Gene delivery is one approach to introduce desired bioactive signals. In this study, we investigated how the properties of MAP hydrogels influence non-viral gene delivery of polyethylenimine-condensed plasmid to cells seeded within the MAP gel. From past studies, we found that gene transfer to cells seeded in tissue culture plastic differed from gene transfer to cells seeded inside hydrogel scaffolds. Since MAP scaffolds are generated from hydrogel microparticles that are approximately 100 µm in diameter, they display local characteristics that can be viewed as two-dimensional or three-dimensional to cells. Thus, we sought to study if gene transfer inside MAP scaffolds differed from gene transfer to cells seeded in tissue culture plastic. We sought to understand the roles of the endocytosis pathway, actin and microtubule dynamics, RhoGTPases, and YAP/TAZ on transfection of human fibroblasts.

It's All in the Delivery: Designing Hydrogels for Cell and Non-viral Gene Therapies.

Hydrogels provide a regenerative medicine platform with their ability to create an environment that supports transplanted or endogenous infiltrating cells and enables these cells to restore or replace the function of tissues lost to disease or trauma. Furthermore, these systems have been employed as delivery vehicles for therapeutic genes, which can direct and/or enhance the function of the transplanted or endogenous cells. Herein, we review recent advances in the development of hydrogels for cell and non-viral gene delivery through understanding the design parameters, including both physical and biological components, on promoting transgene expression, cell engraftment, and ultimately cell function. Furthermore, this review identifies emerging opportunities for combining cell and gene delivery approaches to overcome challenges to the field.

Hydrogels with precisely controlled integrin activation dictate vascular patterning and permeability.

Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here, we show that through engineering hydrogel materials to promote α3/α5ß1 integrin binding, we can promote the formation of a space-filling and mature vasculature compared with hydrogel materials that promote αvß3 integrin binding. In vitro, α3/α5ß1 scaffolds promoted endothelial cells to sprout and branch, forming organized extensive networks that eventually reached and anastomosed with neighbouring branches. In vivo, α3/α5ß1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel formation and non-leaky blood vessels by 10 days post-stroke. In contrast, materials that promote αvß3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF-induced vascular permeability in vivo.

Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks.

Injectable hydrogels can provide a scaffold for in situ tissue regrowth and regeneration, yet gel degradation before tissue reformation limits the gels' ability to provide physical support. Here, we show that this shortcoming can be circumvented through an injectable, interconnected microporous gel scaffold assembled from annealed microgel building blocks whose chemical and physical properties can be tailored by microfluidic fabrication. In vitro, cells incorporated during scaffold formation proliferated and formed extensive three-dimensional networks within 48 h. In vivo, the scaffolds facilitated cell migration that resulted in rapid cutaneous-tissue regeneration and tissue-structure formation within five days. The combination of microporosity and injectability of these annealed gel scaffolds should enable novel routes to tissue regeneration and formation in vivo.

Imine Hydrogels with Tunable Degradability for Tissue Engineering.

A shortage of available organ donors has created a need for engineered tissues. In this context, polymer-based hydrogels that break down inside the body are often used as constructs for growth factors and cells. Herein, we report imine cross-linked gels where degradation is controllable by the introduction of mixed imine cross-links. Specifically, hydrazide-functionalized poly(ethylene glycol) (PEG) reacts with aldehyde-functionalized PEG (PEG-CHO) to form hydrazone linked hydrogels that degrade quickly in media. The time to degradation can be controlled by changing the structure of the hydrazide group or by introducing hydroxylamines to form nonreversible oxime linkages. Hydrogels containing adipohydrazide-functionalized PEG (PEG-ADH) and PEG-CHO were found to degrade more rapidly than gels formed from carbodihydrazide-functionalized PEG (PEG-CDH). Incorporating oxime linkages via aminooxy-functionalized PEG (PEG-AO) into the hydrazone cross-linked gels further stabilized the hydrogels. This imine cross-linking approach should be useful for modulating the degradation characteristics of 3D cell culture supports for controlled cell release.

The chicken chorioallantoic membrane model in biology, medicine and bioengineering.

The chicken chorioallantoic membrane (CAM) is a simple, highly vascularized extraembryonic membrane, which performs multiple functions during embryonic development, including but not restricted to gas exchange. Over the last two decades, interest in the CAM as a robust experimental platform to study blood vessels has been shared by specialists working in bioengineering, development, morphology, biochemistry, transplant biology, cancer research and drug development. The tissue composition and accessibility of the CAM for experimental manipulation, makes it an attractive preclinical in vivo model for drug screening and/or for studies of vascular growth. In this article we provide a detailed review of the use of the CAM to study vascular biology and response of blood vessels to a variety of agonists. We also present distinct cultivation protocols discussing their advantages and limitations and provide a summarized update on the use of the CAM in vascular imaging, drug delivery, pharmacokinetics and toxicology.

Delivery of iPS-NPCs to the Stroke Cavity within a Hyaluronic Acid Matrix Promotes the Differentiation of Transplanted Cells.

Stroke is the leading cause of adult disability with ~80% being ischemic. Stem cell transplantation has been shown to improve functional recovery. However, the overall survival and differentiation of these cells is still low. The infarct cavity is an ideal location for transplantation as it is directly adjacent to the highly plastic peri-infarct region. Direct transplantation of cells near the infarct cavity has resulted in low cell viability. Here we deliver neural progenitor cells derived from induce pluripotent stem cells (iPS-NPC) to the infarct cavity of stroked mice encapsulated in a hyaluronic acid hydrogel matrix to protect the cells. To improve the overall viability of transplanted cells, each step of the transplantation process was optimized. Hydrogel mechanics and cell injection parameters were investigated to determine their effects on the inflammatory response of the brain and cell viability, respectively. Using parameters that balanced the desire to keep surgery invasiveness minimal and cell viability high, iPS-NPCs were transplanted to the stroke cavity of mice encapsulated in buffer or the hydrogel. While the hydrogel did not promote stem cell survival one week post-transplantation, it did promote differentiation of the neural progenitor cells to neuroblasts.

Hybrid photopatterned enzymatic reaction (HyPER) for in situ cell manipulation.

The ability to design artificial extracellular matrices as cell-instructive scaffolds has opened the door to technologies capable of studying the fate of cells in vitro and to guiding tissue repair in vivo. One main component of the design of artificial extracellular matrices is the incorporation of biochemical cues to guide cell phenotype and multicellular organization. The extracellular matrix (ECM) is composed of a heterogeneous mixture of proteins that present a variety of spatially discrete signals to residing cell populations. In contrast, most engineered ECMs do not mimic this heterogeneity. In recent years, photo-deprotection has been used to spatially immobilize signals. However, this approach has been limited mostly to small peptides. Here we combine photo-deprotection with enzymatic reaction to achieve spatially controlled immobilization of active bioactive signals that range from small molecules to large proteins. A peptide substrate for transglutaminase factor XIII (FXIIIa) was caged with a photo-deprotectable group, which was then immobilized to the bulk of a cell-compatible hydrogel. With focused light, the substrate can be deprotected and used to immobilize patterned bioactive signals. This approach offers an innovative strategy to immobilize delicate bioactive signals, such as growth factors, without loss of activity and enables in situ cell manipulation of encapsulated cells.

Identification and analysis of 3D pores in packed particulate materials.

We took the classic 'guess the number of beans in a jar game' and amplified the research question. Rather than estimate the quantity of particles in the jar, we sought to characterize the spaces between them. Here we present an approach for delineating the pockets of empty space (three-dimensional pores) between packed particles, which are hotspots for activity in applications and natural phenomena that deal with particulate materials. We utilize techniques from graph theory to exploit information about particle configuration that allows us to locate important spatial landmarks within the void space. These landmarks are the basis for our pore segmentation, where we consider both interior pores as well as entrance and exit pores into and out of the structure. Our method is robust for particles of varying size, form, stiffness and configuration, which allows us to study and compare three-dimensional pores across a range of packed particle types. We report striking relationships between particles and pores that are described mathematically, and we offer a visual library of pore types. With a meaningful discretization of void space, we demonstrate that packed particles can be understood not by their solid space, but by their empty space.

Nanoenhancer for improving naked DNA electrotransfection In vivo.

Introduction: Electrotransfection (ET) is a non-viral approach widely used for delivery of naked nucleic acids. Its efficiency can be increased in vitro by treatment of cells with various small molecule enhancers. However, these enhancers often fail to improve ET in vivo, presumably due to rapid clearance in tissues after local injection, reducing their cellular uptake. To this end, we propose to develop a new type of ET enhancers, which we term nanoenhancer, that diffuse slowly in tissues and are poorly absorbed by blood and lymph microvessels. Methods: Two nanoenhancers were synthesized with alginate (Alg) and chitosan (Chi) with or without poly (ethylene imine) (PEI). They were used to treat cells in vitro or mouse muscle in the hind leg in vivo prior to ET of plasmid DNA coding reporter genes. At 24 hours post ET, the efficiency of ET was quantified, and compared with that in the untreated controls. Changes in lysosomal size and acidity post nanoenhancer treatment were measured with fluorescence microscopy techniques. Results and discussion: We observed that the pretreatment of cells with the nanoenhancers could enhance the ET efficiency and cell viability in both C2C12 and HCT116 cells in vitro, and the nanoenhancer pretreatment had similar effects on the ET efficiency in vivo. Mechanisms of the enhancement were related to transient inactivation of lysosomal functions triggered by the nanoenhancer treatment. The concept of nanoenhancer will lead to development of new enhancers that can be used to improve ET efficiency in vivo, highlighting its potential in clinical applications.

A Balance between Pro-Inflammatory and Pro-Reparative Macrophages is Observed in Regenerative D-MAPS.

Microporous annealed particle scaffolds (MAPS) are a new class of granular materials generated through the interlinking of tunable microgels, which produce an interconnected network of void space. These microgel building blocks can be designed with different mechanical or bio-active parameters to facilitate cell infiltration and modulate host response. Previously, changing the chirality of the microgel crosslinking peptides from L- to D-amino acids led to significant tissue regeneration and functional recovery in D-MAPS-treated cutaneous wounds. In this study, the immunomodulatory effect of D-MAPS in a subcutaneous implantation model is investigated. How macrophages are the key antigen-presenting cells to uptake and present these biomaterials to the adaptive immune system is uncovered. A robust linker-specific IgG2b/IgG1 response to D-MAPS is detected as early as 14 days post-implantation. The fine balance between pro-regenerative and pro-inflammatory macrophage phenotypes is observed in D-MAPS as an indicator for regenerative scaffolds. The work offers valuable insights into the temporal cellular response to synthetic porous scaffolds and establishes a foundation for further optimization of immunomodulatory pro-regenerative outcomes.

Spatial Confinement Modulates Macrophage Response in Microporous Annealed Particle (MAP) Scaffolds.

Macrophages are essential in the initiation, maintenance, and transition of inflammatory processes such as foreign body response and wound healing. Mounting evidence suggests that physical factors also modulate macrophage activation. 2D in vitro systems demonstrate that constraining macrophages to small areas or channels modulates their phenotypes and changes their responses to known inflammatory agents such as lipopolysaccharide. However, how dimensionality and pore size affect macrophage phenotype is less explored. In this work, the change in macrophage M1/M2 polarization when confined in microporous annealed particle (MAP) scaffolds is studied. Particles sizes (40, 70, and 130 µm) are selected using outputs from software LOVAMAP that analyzes the characteristics of 3D pores in MAP gels. As the size of building block particle correlates with pore size inside the scaffolds, the three  types of scaffold allow us to study how the degree of spatial confinement modulates the behavior of embedded macrophages. Spatially confining macrophages in scaffolds with pore size on the scale of cells leads to a reduced level of the inflammatory response, which is correlated with a change in cell morphology and motility.

Exploring the Role of Spatial Confinement in Immune Cell Recruitment and Regeneration of Skin Wounds.

Microporous annealed particle (MAP) scaffolds are injectable granular materials comprised of micron sized hydrogel particles (microgels). The diameter of these microgels directly determines the size of the interconnected void space between particles where infiltrating or encapsulated cells reside. This tunable porosity allows us to use MAP scaffolds to study the impact of spatial confinement (SC) on both cellular behaviors and the host response to biomaterials. Despite previous studies showing that pore size and SC influence cellular phenotypes, including mitigating the macrophage inflammatory response, there is still a gap in knowledge regarding how SC within a biomaterial modulates immune cell recruitment in vivo in wounds and implants. Thus, we studied the immune cell profile within confined and unconfined biomaterials using small (40 µm), medium (70 µm), and large (130 µm) diameter spherical microgels, respectively. We discovered that MAP scaffolds imparted regenerative wound healing with an IgG1-biased Th2 response. MAP scaffolds generated from 130 µm diameter microgels have a median 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 levels of inflammation.