Magnetic iron oxide (Magnetite, Fe3O4) nanoparticles are widely utilized in magnetic resonance imaging (MRI) and drug delivery applications due to their superparamagnetism. Surface coatings are often employed to change the properties of the magnetite nanoparticles or to modulate their biological responses. In this study, magnetite nanoparticles were fabricated through hydrothermal synthesis. Hydrophobicity is often increased by surface modification with oleic acid. In this study, however, hydrophobicity was introduced through surface modification with n-octyltriethoxysilane. Both the uncoated (hydrophilic) and coated (hydrophobic) individual nanoparticle sizes measured below 20 nm in diameter, a size range in which magnetite nanoparticles exhibit superparamagnetism. Both types of nanoparticles formed aggregates which were characterized by SEM, TEM, and dynamic light scattering (DLS). The coating process significantly increased both individual particle diameter and aggregate sizes. We tested the neurotoxicity of newly synthesized nanoparticles with two mammalian cell lines, PC12 (rat pheochromocytoma) and ReNcell VM (human neural stem cells). Significant differences were observed in cytotoxicity profiles, which suggests that the cell type (rodent versus human) or the presence of serum matters for nanoparticle toxicology studies. Differences in nanoparticle associations/uptake between the two cell types were observed with Prussian Blue staining. Finally, safe concentrations which did not significantly affect neuronal differentiation profiles were identified for further development of the nanoparticles.

Retinoic acid (RA) is a bioactive lipid that has been shown to promote neural stem cell differentiation. However, the highly hydrophobic molecule needs to first solubilize and translocate across the cell membrane in order to exert a biological response. The cell entry of RA can be aided by cell penetrating peptides (CPPs), which are short amino acid sequences that are able to carry bioactive cargo past the cell membrane. In this work, a novel cell penetrating peptide was developed to deliver RA to human neural stem cells and, subsequently, promote neuronal differentiation. The novel CPP consists of a repeating sequence, whose number of repeats is proportional to the efficiency of cell penetration. Using fluorescence microscopy, the mode of translocation was determined to be related to an endocytic pathway. The levels of β-III tubulin (Tubb3) and microtubule associated protein 2 (MAP2) expression in neural stem cells treated with RA conjugated to the CPP were assessed by quantitative immunocytochemistry.

Background: Cartilage progenitor cells (CPCs) are a small but highly proliferative cell population that resides within cartilage. Joint cartilage CPCs have a high chondrogenic potential and superior cartilage formation characteristics; however, CPCs from other cartilage sources more accessible for translation such as ear, nose, and rib are broadly unexplored. Our study illuminates the differences between CPCs from these four cartilages, their corresponding tissue chondrocyte (CC), and bone marrow-derived mesenchymal stem cell (MSC). Methods: CPCs subtypes were isolated from pediatric cartilage via fibronectin selection, immunophenotyped by flow cytometry and compared to MSCs. Trilineage differentiation capacity was assessed via histology and qRT-PCR. Next, triiodothyronine was used to hypertrophically challenge each CPC subset and their corresponding chondrocyte population. After 28 days cartilage pellets were assessed via histology, immunohistochemistry, and qRT-PCR. Findings: Each CPC subset possessed a specific immunophenotypic signature with CD56 as a potential common marker. All CPC subsets proliferated 2-fold faster than MSCs and 4-fold faster than CCs. Additionally, CPCs had a substantially reduced propensity for osteogenic differentiation and very limited adipogenic capacity by histology and gene expression. Finally, all CPC subsets resisted the hypertrophic challenge more than the corresponding chondrocyte population marked by less collagen X secretion and downregulation of hypertrophy associated genes. Interpretation: CPCs represent a promising cell type for cartilage regeneration. The ease of accessibility of the ear and nose CPCs present opportunities for new translational approaches and reduced clinical timelines.

Severe subglottic stenosis develops in over 20,000 infants per year and requires laryngotracheal reconstruction (LTR) to enlarge the airway by implanting autologous cartilage from a rib graft. However, young children often lack sufficiently sized costal cartilage resulting in increased donor site morbidity and operative time, as well as an elevated risk for airway restenosis necessitating revision surgery. To overcome these limitations, we have created a first-of-its-kind scaffold based on porcine meniscal cartilage decellularization (MEND) by selectively digesting the elastin and blood vessels uniquely present in the meniscus to create microchannels that support cellular re-invasion. Here we demonstrated that MEND can be fully recellularized in 3 days with ear-derived cartilage progenitor cells (eCPCs) and reaches structural and functional maturation suitable for implant within 3 weeks of chondrogenic differentiation, a time frame compatible with clinical translation, a first in airway tissue engineering. To further this therapy toward clinical translation, we validated the eCPCs-MEND grafts in a New Zealand white rabbit LTR model. Our results demonstrated airway expansion, graft re-epitheliazation, neocartilage formation, and integration with adjacent native laryngotracheal cartilage, notably at a higher degree than the standard of care of autologous costal cartilage. No instances of adverse events of extrusion, granulation, infection, or calcification were observed in any of the 38 rabbits of our 3 months study. These results demonstrate the feasibility of our translational tissue engineering approach to laryngotracheal reconstruction and could overcome the autograft-associated limitations in pediatric patients and a decrease the risk of invasive revision surgery.