Customized two degree-of-freedom (i.e., compression and torsion) bioreactor to maintain disc explants alive and to apply repeated mechanical loading for two days.

<p> A. close-up view of single station harboring a bovine coccygeal IVD soaked in culture media. Biocompatible materials are enox aluminium (black), polyoxymethylene (POM, white parts) and glass with a “press-fit” design and silicon rings (black rings) to ensure no leakage between glass and POM. <b>B.</b> 4-unit design arranged in 5% CO₂ and 60% humidity incubator. <b>C.</b> Scheme of control of uniaxial compression and axial torsion using fluidic muscle and servo-controlled valve. <b>D.</b> Close-up view of serrated titanium plate surface, which grasps IVD and keeps it in place and ensures nutrition diffusion to the bony endplate.</p

Primers used for quantitative real time PCR in organ culture study.

<p>Primers used for quantitative real time PCR in organ culture study.</p

Relative gene expression after different loading types.

<p>Gene expressions are in log of fold change normalized to control without loading and represented in means ± SEM. SL: Static Loading, DL: Dynamic Loading. * = statistical significant difference with p < 0.05. N = 5 (from 3 animals) compared to control.</p

Relative gene expression after repeated dynamic loading and resting.

<p>Gene expressions are presented in log of fold change normalized to control without loading and represented in means ± SEM. DL: after dynamic loading, DL+Rest: after dynamic loading followed by resting, DL*2: after dynamic loading for 2 days, DL+Rest*2: after dynamic loading followed by resting for two days. N = 5. * = statistical significant difference with p < 0.05. N = 5 (from 5 animals) compared to control.</p

Cell activity and cytoskeleton were maintained after each condition.

<p>Cell activity was measured by resazurin assay (A-B) and cytoskeleton was visualized by f-actin staining (C-J) for NP and AF after each condition. Values are normalized to control without loading and represented in mean ± SEM. Control: Control discs without loading, HS: Heat-Shock, SL: Static Loading, DL: Dynamic loading, DL+Rest: Dynamic loading with Resting, DL*2: dynamic loading for two days, DL+Rest*2: dynamic loading and resting for two days. NP: Nuclues pulposus, AF: Annulus fibrosus. N = 5. RFU: relative fluorescence unit.</p

Encapsulation of Human Spinal Cord Progenitor Cells in Hyaluronan-Gelatin Hydrogel for Spinal Cord Injury Treatment

Transplanting human induced pluripotent stem cells (iPSCs)-derived spinal cord progenitor cells (SCPCs) is a promising approach to treat spinal cord injuries. However, stem cell therapies face challenges in cell survival, cell localization to the targeted site, and the control of cell differentiation. Here, we encapsulated SCPCs in thiol-modified hyaluronan-gelatin hydrogels and optimized scaffold mechanical properties and cell encapsulation density to promote cell viability and neuronal differentiation in vitro and in vivo. Different compositions of hyaluronan-gelatin hydrogels formulated by varying concentrations of poly(ethylene glycol) diacrylate were mechanically characterized by using atomic force microscopy. In vitro SCPC encapsulation study showed higher cell viability and proliferation with lower substrate Young’s modulus (200 Pa vs 580 Pa) and cell density. Moreover, the soft hydrogels facilitated a higher degree of neuronal differentiation with extended filament structures in contrast to clumped cellular morphologies obtained in stiff hydrogels (p in vivo, the optimized SCPC-encapsulated hydrogels resulted in higher cell survival and localization at the transplanted region as compared to cell delivery without hydrogel encapsulation at 2 weeks postimplantation within the rat spinal cord (p < 0.01). Notably, immunostaining demonstrated that the hydrogel-encapsulated SCPCs differentiated along the neuronal and oligodendroglial lineages in vivo. The lack of pluripotency and proliferation also supported the safety of the SCPC transplantation approach. Overall, the injectable hyaluronan-gelatin hydrogel shows promise in supporting the survival and neural differentiation of human SCPCs after transplantation into the spinal cord