Click by Click Microporous Annealed Particle (MAP) Scaffolds.
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ABSTRACT:
SUBMITTER: Darling NJ
PROVIDER: S-EPMC7340246 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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 ?m 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.
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Project description:Intramyocardial injection of hydrogels offers great potential for treating myocardial infarction (MI) in a minimally invasive manner. However, traditional bulk hydrogels generally lack microporous structures to support rapid tissue ingrowth and biochemical signals to prevent fibrotic remodeling toward heart failure. To address such challenges, a novel drug-releasing microporous annealed particle (drugMAP) system is developed by encapsulating hydrophobic drug-loaded nanoparticles into microgel building blocks via microfluidic manufacturing. By modulating nanoparticle hydrophilicity and pregel solution viscosity, drugMAP building blocks are generated with consistent and homogeneous encapsulation of nanoparticles. In addition, the complementary effects of forskolin (F) and Repsox (R) on the functional modulations of cardiomyocytes, fibroblasts, and endothelial cells in vitro are demonstrated. After that, both hydrophobic drugs (F and R) are loaded into drugMAP to generate FR/drugMAP for MI therapy in a rat model. The intramyocardial injection of MAP gel improves left ventricular functions, which are further enhanced by FR/drugMAP treatment with increased angiogenesis and reduced fibrosis and inflammatory response. This drugMAP platform represents a new generation of microgel particles for MI therapy and will have broad applications in regenerative medicine and disease therapy.
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Project description: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.
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Project description:Mimicking growth factor-ECM interactions for promoting cell migration is a powerful technique to improve tissue integration with biomaterial scaffolds for the regeneration of damaged tissues. This has been attempted by scaffold-mediated controlled delivery of exogenous growth factors; however, the predetermined nature of this delivery can limit the scaffold's ability to meet each wound's unique spatiotemporal regenerative needs and presents translational hurdles. To address this limitation, we present a new approach to growth factor presentation by incorporating heparin microislands, which are spatially isolated heparin-containing microparticles that can reorganize and protect endogenous local growth factors via heterogeneous sequestration at the microscale in vitro and result in functional improvements in wound healing. More specifically, we incorporated our heparin microislands within microporous annealed particle (MAP) scaffolds, which allows facile tuning of microenvironment heterogeneity through ratiometric mixing of microparticle sub-populations. In this manuscript, we demonstrate the ability of heparin microislands to heterogeneously sequester applied growth factor and control downstream cell migration in vitro. Further, we present their ability to significantly improve wound healing outcomes (epidermal regeneration and re-vascularization) in a diabetic wound model relative to two clinically relevant controls.
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Project description:Microporous annealed particle (MAP) hydrogel has been a promising scaffold platform technology to promote immediate tissue integration in injured tissue environments. The addition of growth factors has the potential to accelerate tissue integration and enhance scaffold-mediated healing. Growth factor releasing scaffolds face the translational hurdle of limited solubilized protein shelf stability; however, to address this hurdle we present a lyophilized MAP scaffold which can be effectively rehydrated directly prior to use. Our new approach includes a heterogenous MAP scaffold wherein 5% of the microgels contain immobilized heparin loaded with epidermal growth factor (EGF) at 1 μg mL-1. We demonstrate that these scaffolds, which are directly loaded with EGF following lyophilization maintain equivalent properties to scaffolds loaded passively via diffusion into water-swollen microgels, including EGF release profiles and cell migration studies that did not significantly differ. Further, these heterogeneous scaffolds exhibit a significant increase in cellular migration in vitro and quicker re-epithelialization in vivo. This progress on spatially heterogenous growth factor release from MAP scaffolds has great potential to improve complex wound treatment and advance the field of growth factor releasing scaffolds.
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Project description:Microporous annealed particle (MAP) hydrogels are an attractive platform for engineering biomaterials with controlled heterogeneity. Here, we introduce a microfluidic method to create physicochemical gradients within poly(ethylene glycol) based MAP hydrogels. By combining microfluidic mixing and droplet generator modules, microgels with varying properties were produced by adjusting the relative flow rates between two precursor solutions and collected layer-by-layer in a syringe. Subsequently, the microgels were injected out of the syringe and then annealed with thiol-ene click chemistry. Fluorescence intensity measurements of constructs annealed in vitro and after mock implantation into a tissue defect showed that a continuous gradient profile was achieved and maintained after injection, indicating utility for in situ hydrogel formation. The effects of physicochemical property gradients on human mesenchymal stem cells (hMSCs) were also studied. Microgel stiffness was studied first, and the hMSCs exhibited increased spreading and proliferation as stiffness increased along the gradient. Microgel degradability was also studied, revealing a critical degradability threshold above which the hMSCs spread robustly and below which they were isolated and exhibited reduced spreading. This method of generating spatial gradients in MAP hydrogels could be further used to gain new insights into cell-material interactions, which could be leveraged for tissue engineering applications. A new droplet microfluidic approach to obtain microporous annealed particle hydrogels with physicochemical gradients is presented. Gradient formation is achieved by precisely controlling the mixing of two precursor solutions, and the gradient can be maintained after injection. This approach can be leveraged to produce new materials for tissue repair and to gain unique insights on cell-material interactions.
| S-EPMC10783553 | biostudies-literature
Project description:Inter-particle secondary crosslinks allow microporous annealed particle (MAP) hydrogels to be formed. Methods to introduce secondary crosslinking networks in MAP hydrogels include particle jamming, annealing with covalent bonds, and reversible noncovalent interactions. Here, we investigate the effect of two different approaches to secondary crosslinking of polyethylene glycol (PEG) microgels via reversible guest-host interactions. We generated a dual-particle MAP-PEG hydrogel using two species of PEG microgels, one functionalized with the guest molecule, adamantane, and the other with the host molecule, β-cyclodextrin (Inter-MAP-PEG). In a different approach, a mono-particle MAP-PEG hydrogel was generated using one species of microgel functionalized with both guest and host molecules (Intra-MAP-PEG). The Intra-MAP-PEG formed a homogenous distribution due to the single type of microgels used. We then compared the mechanical properties of these two types of MAP-PEG hydrogels and found that Intra-MAP-PEG resulted in significantly softer gels with lower yield stress. We investigated the effect of intra-particle guest-host interactions through titrated weight percentage and the concentration of functional groups added to the hydrogel. We found that there was an ideal concentration of guest-host molecules that enables intra- and inter-particle guest-host interactions with sufficient covalent crosslinking. Based on these studies, Intra-MAP-PEG provides a homogeneous guest-host hydrogel that is shear-thinning with reversible secondary crosslinking.
| S-EPMC10909382 | biostudies-literature
Project description:Designing scaffolds for polyplex-mediated therapeutic gene delivery has a number of applications in regenerative medicine, such as for tissue repair after wounding or disease. Microporous annealed particle (MAP) hydrogels are an emerging class of porous biomaterials, formed by annealing microgel particles to one another in situ to form a porous bulk scaffold. MAP gels have previously been shown to support and enhance proliferative and regenerative behaviors both in vitro and in vivo. Therefore, coupling gene delivery with MAP hydrogels presents a promising approach for therapy development. To optimize MAP hydrogels for gene delivery, we studied the effects of particle size and stiffness as well as adhesion potential on cell surface area and proliferation and then correlated this information with the ability of cells to become transfected while seeded in these scaffolds. We find that the void space size as well as the presentation of integrin ligands influence transfection efficiency. This work demonstrates the importance of considering MAP material properties for guiding cell spreading, proliferation, and gene transfer. STATEMENT OF SIGNIFICANCE: Microporous annealed particle (MAP) hydrogels are an emerging class of porous biomaterials, formed by annealing spherical microgels together in situ, creating a porous scaffold from voids between the packed beads. Here we investigated the effects of MAP physical and adhesion properties on cell spreading, proliferation, and gene transfer in fibroblasts. Particle size and void space influenced spreading and proliferation, with larger particles improving transfection. MAP stiffness was also important, with stiffer scaffolds increasing proliferation, spreading, and transfection, contrasting studies in nonporous hydrogels that showed an inverse response. Last, RGD ligand concentration and presentation modulated spreading similar to non-MAP hydrogels. These findings reveal relationships between MAP properties and cell processes, suggesting how MAP can be tuned to improve future design approaches.
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Project description:Microporous annealed particle (MAP) hydrogels are promising materials for delivering therapeutic cells. It has previously been shown that spreading and mechanosensing activation of human mesenchymal stem cells (hMSCs) incorporated in these materials can be modulated by tuning the modulus of the microgel particle building blocks. However, the effects of degradability and functionalization with different integrin-binding peptides on cellular responses has not been explored. In this work, RGDS functionalized and enzymatically degradable poly(ethylene glycol) (PEG) microgels were annealed into MAP hydrogels via thiol-ene click chemistry and photopolymerization. During cell-mediated degradation, the microgel surfaces were remodeled to wrinkles or ridges, but the scaffold integrity was maintained. Moreover, cell spreading, proliferation, and secretion of extracellular matrix proteins were significantly enhanced in faster matrix metalloproteinase degrading (KCGPQGIWGQCK) MAP hydrogels compared to non-degradable controls after 8 days of culture. We subsequently evaluated paracrine activity by hMSCs seeded in the MAP hydrogels functionalized with either RGDS or c(RRETAWA), which is specific for ?5?1 integrins, and evaluated the interplay between degradability and integrin-mediated signaling. Importantly, c(RRETAWA) functionalization upregulated secretion of bone morphogenetic protein-2 overall and on a per cell basis, but this effect was critically dependent on microgel degradability. In contrast, RGDS functionalization led to higher overall vascular endothelial growth factor secretion in degradable scaffolds due to the high cell number. These results demonstrate that integrin-binding peptides can modulate hMSC behavior in PEG-based MAP hydrogels, but the results strongly depend on the susceptibility of the microgel building blocks to cell-mediated matrix remodeling. This relationship should be considered in future studies aiming to further develop these materials for stem cell delivery and tissue engineering applications. STATEMENT OF SIGNIFICANCE: Microporous annealed particle (MAP) hydrogels are attracting increasing interest for tissue repair and regeneration and have shown superior results compared to conventional hydrogels in multiple applications. Here, we studied the impact of MAP hydrogel degradability and functionalization with different integrin-binding peptides on human mesenchymal stem cells (hMSCs) that were incorporated during particle annealing. Degradability was found to improve cell growth, spreading, and extracellular matrix production regardless of the integrin-binding peptide. Moreover, in degradable MAP hydrogels the integrin-binding peptide c(RRETAWA) was found to increase osteogenic protein expression by hMSCs compared to RGDS-functionalized MAP hydrogels. These results have important implications for the development of a MAP hydrogel-based hMSC delivery system for bone tissue engineering.
| S-EPMC6960331 | biostudies-literature
Project description:The design of novel biomaterials for regenerative medicine requires incorporation of well-defined physical and chemical properties that mimic the native extracellular matrix (ECM). Here, we report the synthesis and characterization of porous foams prepared by high internal phase emulsion (HIPE) templating using amphiphilic copolymers that act as surfactants during the HIPE process. We combine different copolymers exploiting oil-water interface confined phase separation to engineer the surface topology of foam pores with nanoscopic domains of cell inert and active chemistries mimicking native matrix. We further demonstrate how proteins and hMSCs adhere in a domain specific manner.
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