Conductive Microgel Annealed Scaffolds Enhance Myogenic Potential of Myoblastic Cells.

Conductive biomaterials may capture native or exogenous bioelectric signaling, but incorporation of conductive moieties is limited by cytotoxicity, poor injectability, or insufficient stimulation. Microgel annealed scaffolds are promising as hydrogel-based materials due to their inherent void space that facilitates cell migration and proliferation better than nanoporous bulk hydrogels. Conductive microgels are generated from poly(ethylene) glycol (PEG and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT: PSS) to explore the interplay of void volume and conductivity on myogenic differentiation. PEDOT: PSS increases microgel conductivity two-fold while maintaining stiffness, annealing strength, and viability of associated myoblastic cells. C2C12 myoblasts exhibit increases in the late-stage differentiation marker myosin heavy chain as a function of both porosity and conductivity. Myogenin, an earlier marker, is influenced only by porosity. Human skeletal muscle-derived cells exhibit increased Myod1, insulin like growth factor-1, and insulin-like growth factor binding protein 2 at earlier time points on conductive microgel scaffolds compared to non-conductive scaffolds. They also secrete more vascular endothelial growth factor at early time points and express factors that led to macrophage polarization patterns observe during muscle repair. These data indicate that conductivity aids myogenic differentiation of myogenic cell lines and primary cells, motivating the need for future translational studies to promote muscle repair.

Conductive microgel annealed scaffolds enhance myogenic potential of myoblastic cells.

Bioelectricity is an understudied phenomenon to guide tissue homeostasis and regeneration. Conductive biomaterials may capture native or exogenous bioelectric signaling, but incorporation of conductive moieties is limited by cytotoxicity, poor injectability, or insufficient stimulation. Microgel annealed scaffolds are promising as hydrogel-based materials due to their inherent void space that facilitates cell migration and proliferation better than nanoporous bulk hydrogels. We generated conductive microgels from poly(ethylene) glycol and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) to explore the interplay of void volume and conductivity on myogenic differentiation. PEDOT:PSS increased microgel conductivity over 2-fold while maintaining stiffness, annealing strength, and viability of associated myoblastic cells. C2C12 myoblasts exhibited increases in the late-stage differentiation marker myosin heavy chain as a function of both porosity and conductivity. Myogenin, an earlier marker, was influenced only by porosity. Human skeletal muscle derived cells exhibited increased Myod1 , IGF-1, and IGFBP-2 at earlier timepoints on conductive microgel scaffolds compared to non-conductive scaffolds. They also secreted higher levels of VEGF at early timepoints and expressed factors that led to macrophage polarization patterns observed during muscle repair. These data indicate that conductivity aids myogenic differentiation of myogenic cell lines and primary cells, motivating the need for future translational studies to promote muscle repair.

Electroconductive agarose hydrogels modulate mesenchymal stromal cell adhesion and spreading through protein adsorption.

Electrically conductive biomaterials direct cell behavior by capitalizing on the effect of bioelectricity in tissue homeostasis and healing. Many studies have leveraged conductive biomaterials to influence cells and improve tissue healing, even in the absence of external stimulation. However, most studies using electroactive materials neglect characterizing how the inclusion of conductive additives affects the material's mechanical properties, and the interplay between substrate electrical and mechanical properties on cell behavior is poorly understood. Furthermore, mechanisms dictating how electrically conductive materials affect cell behavior in the absence of external stimulation are not explicit. In this study, we developed a mechanically and electrically tunable conductive hydrogel using agarose and the conductive polymer PEDOT:PSS. Under certain conditions, we observed that the hydrogel physical and electrical properties were decoupled. We then seeded human mesenchymal stromal cells (MSCs) onto the hydrogels and observed enhanced adhesion and spreading of MSCs on conductive substrates, regardless of the hydrogel mechanical properties, and despite the gels having no cell-binding sites. To explain this observation, we measured protein interaction with the gels and found that charged proteins adsorbed significantly more to conductive hydrogels. These data demonstrate that conductivity promotes cell adhesion, likely by facilitating increased adsorption of proteins associated with cell binding, providing a better understanding of the mechanism of action of electrically conductive materials.

Mesenchymal stromal cell spheroids in sulfated alginate enhance muscle regeneration.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is critically linked to the potency of the complex mixture of growth factors, cytokines, exosomes, and other biological cues that they secrete. The duration of cell-based approaches is limited by rapid loss of cells upon implantation, motivating the need to prolong cell viability and extend the therapeutic influence of the secretome. We and others demonstrated that the secretome is upregulated when MSCs are formed into spheroids. Although the efficacy of the MSC secretome has been characterized in the literature, no studies have reported the therapeutic benefit of in situ sequestration of the secretome within a wound site using engineered biomaterials. We previously demonstrated the capacity of sulfated alginate hydrogels to sequester components of the MSC secretome for prolonged presentation in vitro, yet the efficacy of this platform has not been evaluated in vivo. In this study, we used sulfated alginate hydrogels loaded with MSC spheroids to aid in the regeneration of a rat muscle crush injury. We hypothesized that the use of sulfated alginate to bind therapeutically relevant growth factors from the MSC spheroid secretome would enhance muscle regeneration by recruiting host cells into the tissue site. The combination of sulfated alginate and MSC spheroids resulted in decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions 2 weeks after injury. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreased fibrosis and increased functional regeneration of muscle. STATEMENT OF SIGNIFICANCE: The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is attributed to the complex diversity of the secretome. Cell-based approaches are limited by rapid cell death, motivating the need to extend the availability of the secretome. We previously demonstrated that sulfated alginate hydrogels sequester components of the MSC secretome for prolonged presentation in vitro, yet no studies have reported the in situ sequestration of the secretome. Herein, we transplanted MSC spheroids in sulfated alginate hydrogels to promote muscle regeneration. MSC spheroids in sulfated alginate decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreasing fibrosis and increasing functional muscle regeneration.

Endogenous Electric Signaling as a Blueprint for Conductive Materials in Tissue Engineering.

Bioelectricity plays an important role in cell behavior and tissue modulation, but is understudied in tissue engineering research. Endogenous electrical signaling arises from the transmembrane potential inherent to all cells and contributes to many cell behaviors, including migration, adhesion, proliferation, and differentiation. Electrical signals are also involved in tissue development and repair. Synthetic and natural conductive materials are under investigation for leveraging endogenous electrical signaling cues in tissue engineering applications due to their ability to direct cell differentiation, aid in maturing electroactive cell types, and promote tissue functionality. In this review, we provide a brief overview of bioelectricity and its impact on cell behavior, report recent literature using conductive materials for tissue engineering, and discuss opportunities within the field to improve experimental design when using conductive substrates.

Sulfated Alginate Hydrogels Prolong the Therapeutic Potential of MSC Spheroids by Sequestering the Secretome.

Cell-based approaches to tissue repair suffer from rapid cell death upon implantation, limiting the window for therapeutic intervention. Despite robust lineage-specific differentiation potential in vitro, the function of transplanted mesenchymal stromal cells (MSCs) in vivo is largely attributed to their potent secretome comprising a variety of growth factors (GFs). Furthermore, GF secretion is markedly increased when MSCs are formed into spheroids. Native GFs are sequestered within the extracellular matrix (ECM) via sulfated glycosaminoglycans, increasing the potency of GF signaling compared to their unbound form. To address the critical need to prolong the efficacy of transplanted cells, alginate hydrogels are modified with sulfate groups to sequester endogenous heparin-binding GFs secreted by MSC spheroids. The influence of crosslinking method and alginate modification is assessed on mechanical properties, degradation rate, and degree of sulfate modification. Sulfated alginate hydrogels sequester a mixture of MSC-secreted endogenous biomolecules, thereby prolonging the therapeutic effect of MSC spheroids for tissue regeneration. GFs are sequestered for longer durations within sulfated hydrogels and retain their bioactivity to regulate endothelial cell tubulogenesis and myoblast infiltration. This platform has the potential to prolong the therapeutic benefit of the MSC secretome and serve as a valuable tool for investigating GF sequestration.

Proangiogenic Collagen-Binding Glycan Therapeutic Promotes Endothelial Cell Angiogenesis.

Stimulating angiogenesis during wound healing continues to present a significant clinical challenge, given the limitations of current strategies to maintain therapeutic doses of growth factors and endothelial cell efficacy. Incorporating a balance of specific cues to encourage endothelial cell engraftment and cytokines to facilitate angiogenesis is necessary for blood vessel growth in the proinflammatory wound environment. Here, we incorporate a previously designed peptide (LXW7) capable of binding to the αvß3 integrin of endothelial cells with a dermatan sulfate glycosaminoglycan backbone grafted with collagen-binding peptides (SILY). By exploiting αvß3 integrin-mediated VEGF signaling, we propose an alternative strategy to overcome shortcomings of traditional growth factor therapy while homing the peptide to the wound bed. In this study, we describe the synthesis and optimization of LXW7-DS-SILY (LDS) variants and evaluate their angiogenic potential in vitro and in vivo. LDS displayed binding to collagen and endothelial cells. In vitro, the LDS variant with six LXW7 peptides increased endothelial cell proliferation, migration, and tubule formation through increased VEGFR2 phosphorylation compared to nontreated controls. In an in vivo chick chorioallantoic membrane assay, LDS laden collagen hydrogels increased blood vessel formation by 43% in comparison to the organism matched blank hydrogels. Overall, these findings demonstrate the potential of a robust targeted glycan therapeutic for promoting angiogenesis during wound healing.