Neurogenic Cell Behavior in 3D Culture Enhanced Within a Highly Compliant Synthetic Hydrogel Platform Formed via Competitive Crosslinking.

Purpose: Scaffold materials that better support neurogenesis are still needed to improve cell therapy outcomes for neural tissue damage. We have used a modularly tunable, highly compliant, degradable hydrogel to explore the impacts of hydrogel compliance stiffness on neural differentiation. Here we implemented competitive matrix crosslinking mechanics to finely tune synthetic hydrogel moduli within soft tissue stiffnesses, a range much softer than typically achievable in synthetic crosslinked hydrogels, providing a modularly controlled and ultrasoft 3D culture model which supports and enhances neurogenic cell behavior. Methods: Soluble competitive allyl monomers were mixed with proteolytically-degradable poly(ethylene glycol) diacrylate derivatives and crosslinked to form a matrix, and resultant hydrogel stiffness and diffusive properties were evaluated. Neural PC12 cells or primary rat fetal neural stem cells (NSCs) were encapsulated within the hydrogels, and cell morphology and phenotype were investigated to understand cell-matrix interactions and the effects of environmental stiffness on neural cell behavior within this model. Results: Addition of allyl monomers caused a concentration-dependent decrease in hydrogel compressive modulus from 4.40 kPa to 0.26 kPa (natural neural tissue stiffness) without influencing soluble protein diffusion kinetics through the gel matrix. PC12 cells encapsulated in the softest hydrogels showed significantly enhanced neurite extension in comparison to PC12s in all other hydrogel stiffnesses tested. Encapsulated neural stem cells demonstrated significantly greater spreading and elongation in 0.26 kPa alloc hydrogels than in 4.4 kPa hydrogels. When soluble growth factor deprivation (for promotion of neural differentiation) was evaluated within the neural stiffness gels (0.26 kPa), NSCs showed increased neuronal marker expression, indicating early enhancement of neurogenic differentiation. Conclusions: Implementing allyl-acrylate crosslinking competition reduced synthetic hydrogel stiffness to provide a supportive environment for 3D neural tissue culture, resulting in enhanced neurogenic behavior of encapsulated cells. These results indicate the potential suitability of this ultrasoft hydrogel system as a model platform for further investigating environmental factors on neural cell behavior. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-024-00794-2.

Microbubble-Nanoparticle Complexes for Ultrasound-Enhanced Cargo Delivery.

Targeted delivery of therapeutics to specific tissues is critically important for reducing systemic toxicity and optimizing therapeutic efficacy, especially in the case of cytotoxic drugs. Many strategies currently exist for targeting systemically administered drugs, and ultrasound-controlled targeting is a rapidly advancing strategy for externally-stimulated drug delivery. In this non-invasive method, ultrasound waves penetrate through tissue and stimulate gas-filled microbubbles, resulting in bubble rupture and biophysical effects that power delivery of attached cargo to surrounding cells. Drug delivery capabilities from ultrasound-sensitive microbubbles are greatly expanded when nanocarrier particles are attached to the bubble surface, and cargo loading is determined by the physicochemical properties of the nanoparticles. This review serves to highlight and discuss current microbubble-nanoparticle complex component materials and designs for ultrasound-mediated drug delivery. Nanocarriers that have been complexed with microbubbles for drug delivery include lipid-based, polymeric, lipid-polymer hybrid, protein, and inorganic nanoparticles. Several schemes exist for linking nanoparticles to microbubbles for efficient nanoparticle delivery, including biotin-avidin bridging, electrostatic bonding, and covalent linkages. When compared to unstimulated delivery, ultrasound-mediated cargo delivery enables enhanced cell uptake and accumulation of cargo in target organs and can result in improved therapeutic outcomes. These ultrasound-responsive delivery complexes can also be designed to facilitate other methods of targeting, including bioactive targeting ligands and responsivity to light or magnetic fields, and multi-level targeting can enhance therapeutic efficacy. Microbubble-nanoparticle complexes present a versatile platform for controlled drug delivery via ultrasound, allowing for enhanced tissue penetration and minimally invasive therapy. Future perspectives for application of this platform are also discussed in this review.

Adding Dynamic Biomolecule Signaling to Hydrogel Systems via Tethered Photolabile Cell-Adhesive Proteins.

Sequential biochemical signaling events direct key native tissue processes including disease progression, wound healing and angiogenesis, and tissue regeneration. While in vitro modeling of these processes is critical to understanding endogenous tissue behavior and improving therapeutic outcomes, current models inadequately recapitulate the dynamism of these signaling events. Even the most advanced current synthetic tissue culture constructs are restricted in their capability to sequentially add and remove the same molecule to model transient signaling. Here, we developed a genetically encoded method for reversible biochemical signaling within poly(ethylene glycol) (PEG)-based hydrogels for greater accuracy of modeling tissue regeneration within a reductionist environment. We designed and implemented a recombinant protein with a SpyCatcher domain connected to a cell-adhesive RGDS peptide domain by a light-cleavable domain known as PhoCl. This protein was shown to bind to SpyTag-functionalized PEG-matrices via SpyTag-SpyCatcher isopeptide bonding to present RGDS adhesive ligands to cells. Upon 405 nm light exposure, the PhoCl domain was cleaved to subsequently release the RGDS peptide, which diffused out of the matrix. This system was implemented to confer reversible adhesion of 3T3 fibroblasts to the PEG-based hydrogel surface in 2D culture (73.36 ± 21.47% cell removal upon cell-compatible light exposure) and temporal control over cell spreading over time in 3D culture within cell-degradable PEG-based hydrogels, demonstrating the capability of this system to present dynamic signaling events to cells toward modeling native tissue processes within in a controlled, ECM-mimetic matrix.

Induction of Neurogenesis and Angiogenesis in a Rat Hemisection Spinal Cord Injury Model With Combined Neural Stem Cell, Endothelial Progenitor Cell, and Biomimetic Hydrogel Matrix Therapy.

Acute spinal cord injury is a devastating injury that may lead to loss of independent function. Stem-cell therapies have shown promise; however, a clinically efficacious stem-cell therapy has yet to be developed. Functionally, endothelial progenitor cells induce angiogenesis, and neural stem cells induce neurogenesis. In this study, we explored using a multimodal therapy combining endothelial progenitor cells with neural stem cells encapsulated in a bioactive biomimetic hydrogel matrix to facilitate stem cell-induced neurogenesis and angiogenesis in a rat hemisection spinal cord injury model. DESIGN: Laboratory experimentation. SETTING: University laboratory. SUBJECTS: Female Fischer 344 rats. INTERVENTIONS: Three groups of rats: 1) control, 2) biomimetic hydrogel therapy, and 3) combined neural stem cell, endothelial progenitor cell, biomimetic hydrogel therapy underwent right-sided spinal cord hemisection at T9-T10. The blinded Basso, Beattie, and Bresnahan motor score was obtained weekly; after 4 weeks, observational histologic analysis of the injured spinal cords was completed. MEASUREMENTS AND MAIN RESULTS: Blinded Basso, Beattie, and Bresnahan motor score of the hind limb revealed significantly improved motor function in rats treated with combined neural stem cell, endothelial progenitor cell, and biomimetic hydrogel therapy (p < 0.05) compared with the control group. The acellular biomimetic hydrogel group did not demonstrate a significant improvement in motor function compared with the control group. Immunohistochemistry evaluation of the injured spinal cords demonstrated de novo neurogenesis and angiogenesis in the combined neural stem cell, endothelial progenitor cell, and biomimetic hydrogel therapy group, whereas, in the control group, a gap or scar was found in the injured spinal cord. CONCLUSIONS: This study demonstrates proof of concept that multimodal therapy with endothelial progenitor cells and neural stem cells combined with a bioactive biomimetic hydrogel can be used to induce de novo CNS tissue in an injured rat spinal cord.

Modulating Functionalized Poly(ethylene glycol) Diacrylate Hydrogel Mechanical Properties through Competitive Crosslinking Mechanics for Soft Tissue Applications.

Local mechanical stiffness influences cell behavior, and thus cell culture scaffolds should approximate the stiffness of the tissue type from which the cells are derived. In synthetic hydrogels, this has been difficult to achieve for very soft tissues such as neural. This work presents a method for reducing the stiffness of mechanically and biochemically tunable synthetic poly(ethylene glycol) diacrylate hydrogels to within the soft tissue stiffness regime by altering the organization of the crosslinking sites. A soluble allyl-presenting monomer, which has a higher propensity for chain termination than acrylate monomers, was introduced into the PEG-diacrylate hydrogel precursor solution before crosslinking, resulting in acrylate-allyl competition and a reduction in gel compressive modulus from 5.1 ± 0.48 kPa to 0.32 ± 0.09 kPa. Both allyl monomer concentration and chemical structure were shown to influence the effectiveness of competition and change in stiffness. Fibroblast cells demonstrated a 37% reduction in average cell spread area on the softest hydrogels produced as compared to cells on control hydrogels, while the average percentage of neural cells extending neurites increased by 41% on these hydrogels, demonstrating the potential for this technology to serve as a soft tissue culture system.

Copper-encapsulated vertically aligned carbon nanotube arrays.

A new procedure is described for the fabrication of vertically aligned carbon nanotubes (VACNTs) that are decorated, and even completely encapsulated, by a dense network of copper nanoparticles. The process involves the conformal deposition of pyrolytic carbon (Py-C) to stabilize the aligned carbon-nanotube structure during processing. The stabilized arrays are mildly functionalized using oxygen plasma treatment to improve wettability, and they are then infiltrated with an aqueous, supersaturated Cu salt solution. Once dried, the salt forms a stabilizing crystal network throughout the array. After calcination and H2 reduction, Cu nanoparticles are left decorating the CNT surfaces. Studies were carried out to determine the optimal processing parameters to maximize Cu content in the composite. These included the duration of Py-C deposition and system process pressure as well as the implementation of subsequent and multiple Cu salt solution infiltrations. The optimized procedure yielded a nanoscale hybrid material where the anisotropic alignment from the VACNT array was preserved, and the mass of the stabilized arrays was increased by over 24-fold because of the addition of Cu. The procedure has been adapted for other Cu salts and can also be used for other metal salts altogether, including Ni, Co, Fe, and Ag. The resulting composite is ideally suited for application in thermal management devices because of its low density, mechanical integrity, and potentially high thermal conductivity. Additionally, further processing of the material via pressing and sintering can yield consolidated, dense bulk composites.