Though crucial for natural bone healing, local calcium ion (Ca²⁺) and phosphate ion (P_i) concentrations can exceed the cytotoxic limit leading to mitochondrial overload, oxidative stress, and cell death. For bone tissue engineering applications, H₂S can be employed as a cytoprotective molecule to enhance mesenchymal stem cell (MSC) tolerance to cytotoxic Ca²⁺/P_i concentrations. Varied concentrations of sodium hydrogen sulfide (NaSH), a fast-releasing H₂S donor, were applied to assess the influence of H₂S on MSC proliferation. The results suggested a toxicity limit of 4 mM for NaSH and that 1 mM of NaSH could improve cell proliferation and differentiation in the presence of cytotoxic levels of Ca²⁺ (32 mM) and/or P_i (16 mM). To controllably deliver H₂S over time, a novel donor molecule (thioglutamic acid—GluSH) was synthesized and evaluated for its H₂S release profile. Excitingly, GluSH successfully maintained cytoprotective level of H₂S over 7 days. Furthermore, MSCs exposed to cytotoxic Ca²⁺/P_i concentrations in the presence of GluSH were able to thrive and differentiate into osteoblasts. These findings suggest that the incorporation of a sustained H₂S donor such as GluSH into CaP-based bone graft substitutes can facilitate considerable cytoprotection, making it an attractive option for complex bone regenerative engineering applications.

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. Funding CHOP Research Institute, Frontier Program in Airway Disorders of CHOP, NIH (R21HL159521), NSF-GRFP (DGE-1845298)

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.

Severe subglottic stenosis develops in over 20,000 infants per year and requires laryngotracheal reconstruction to enlarge the airway by implanting autologous cartilage from a rib graft. However, young children often lack sufficiently sized costal cartilage for this procedure, 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 create a scaffold based on porcine meniscal cartilage decellularization – which we term MEND – by selectively digesting away the elastin and blood vessels uniquely present in the meniscus; this creates microchannels that support cellular re-invasion. Here we demonstrate that MEND can be fully recellularized in 3 days with ear-derived cartilage progenitor cells and reaches structural and functional maturation suitable for implant within 3 weeks of chondrogenic differentiation, a time frame compatible with clinical translation. To further this therapy toward clinical translation, we validate the ear-derived cartilage progenitor cell-MEND grafts in a New Zealand white rabbit laryngotracheal reconstruction model. Our results demonstrate airway expansion, graft re-epithelialization, neocartilage formation, and integration with adjacent native laryngotracheal cartilage at 3 months. Notably, MEND implants perform better in all outcomes than autologous costal cartilage, the standard of care. No instances of adverse events such as extrusion, granulation, infection, or calcification are observed in any of the 38 rabbits in our 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, decreasing the need for invasive revision surgery.