Gergely, C. | bioRxiv Science

bioRxiv2024

Transdifferentiation of Human Dental Pulp Mesenchymal Stem Cells into Spiral Ganglion-like Neurons

Spiral ganglion neurons (SGN) carry auditory information from sensory hair cells (HCs) to the brain. These auditory neurons, which are the target neurons of cochlear implants, degenerate following sensorineural hearing loss (SNHL). Prosthetic devices such as cochlear implants function by bypassing lost HCs and stimulating the residual SGNs, allowing restoration of hearing in deaf patients. Emerging cell-replacement therapies for SNHL include replacing damaged SGNs using stem cell-derived otic neuronal progenitors (ONPs). However, the availability of renewable, accessible, and patient-matched sources of human stem cells constitutes a major prerequisite towards cell replacement for auditory nerve recovery. Human dental pulp stem cells (hDPSCs) extracted from human wisdom teeth are self-renewing stem cells that originate from the neural crest during development. In this study, we developed a stepwise in vitro guidance procedure to differentiate hDPSCs into ONPs and then to SGNs. The procedure relies on the modulation of BMP and TGF-{beta} pathways for neurosphere formation as a first step, then a differentiation step based on two culture paradigms exploiting major signaling pathways (Wnt, Shh, RA) and neurotrophic factors involved in early otic neurogenesis. Gene and protein expression analyses revealed efficient induction of a comprehensive panel of known ONP and SGN-like cell markers over the course of in vitro differentiation. The use of atomic force microscopy revealed that hDPSC-derived SGN-like cells exhibit similar nanomechanical properties compared to their in vivo SGN counterparts. Furthermore, neurites extended between hDPSC-derived ONPs and rat SGN explants 4-6 days after co-culturing, suggesting the formation of neuronal contacts. These data indicate that the in vitro differentiated cells closely replicate the phenotypic and nanomechanical characteristics of human SGNs, advancing our culture differentiation system to the level to be used in next-generation cochlear implants and/or inner ear cell-based strategies for SNHL.

bioengineering↗

bioRxiv2023

Volumetric Printing across Melt Electrowritten Scaffolds Fabricates Multi-Material Living Constructs with Tunable Architecture and Mechanics

Major challenges in biofabrication revolve around capturing the complex, hierarchical composition of native tissues. However, individual 3D printing techniques have limited capacity to produce composite biomaterials with multi-scale resolution. Volumetric bioprinting recently emerged as a paradigm-shift in biofabrication. This ultra-fast, light-based technique sculpts cell-laden hydrogel bioresins into three-dimensional structures in a layerless fashion, providing unparalleled design freedom over conventional bioprinting. However, it yields prints with low mechanical stability, since soft, cell-friendly hydrogels are used. Herein, for the first time, the possibility to converge volumetric bioprinting with melt electrowriting, which excels at patterning microfibers, is shown for the fabrication of tubular hydrogel-based composites with enhanced mechanical behavior. Despite including non-transparent melt electrowritten scaffolds into the volumetric printing process, high-resolution bioprinted structures were successfully achieved. Tensile, burst and bending mechanical properties of printed tubes were tuned altering the electrowritten mesh design, resulting in complex, multi-material tubular constructs with customizable, anisotropic geometries that better mimic intricate biological tubular structures. As a proof-of-concept, engineered vessel-like structures were obtained by building tri-layered cell-laden vessels, and features (valves, branches, fenestrations) that could be resolved only by synergizing these printing methods. This multi-technology convergence offers a new toolbox for manufacturing hierarchical and mechanically tunable multi-material living structures.

bioengineering↗