Investigating Mechanisms of Subcutaneous Preconditioning Incubation for Neural Stem Cell Embedded Hydrogels

Abstract

Stem cells are a vital component of regenerative medicine therapies, however, only a fraction of stem cells delivered to the central nervous system following injury survive the inflammatory environment. Previously, we showed that subcutaneous preconditioning of neural stem cell (NSC) embedded hydrogels for 28 days improved spinal cord injury (SCI) functional outcomes over controls. Here, we investigated the mechanism of subcutaneous preconditioning of NSC-embedded hydrogels, with and without the known neurogenic cue, interferon gamma (IFN-γ), for 3, 14, or 28 days to refine and identify subcutaneous preconditioning conditions by measurement of neurogenic markers and cytokines. Studying the preconditioning mechanism, we found that subcutaneous foreign body response (FBR) associated cytokines infiltrated the scaffold in groups with and without NSCs, with time point effects. A pro-inflammatory environment with upregulated interleukin (IL)-6, IL-10, macrophage inflammatory protein (MIP)-1, MIP-2, IFN-γ-inducible protein 10 (IP-10), tumor necrosis factor-α (TNF-α), and IL-12p70 was observed on day 3. By 14 and 28 days, there was an increase in pro-regenerative cytokines (IL-13, IL-4) along with pro-inflammatory markers IL-1β, IP-10, and RANTES (regulated on activation, normal T cell expressed, and secreted) potentially part of the mechanism that had an increased functional outcome in SCI. Coinciding with changes in cytokines, the macrophage population increased over time from 3 to 28 days, whereas neutrophils peaked at 3 days with a significant decrease at later time points. Expression of the neuronal marker βIII tubulin in differentiating NSCs was supported at 3 days in the presence of soluble and immobilized IFN-γ and at 14 days by immobilized IFN-γ only, but it was greatly attenuated in all conditions at 28 days, partially because of dilution via host cell infiltration. We conclude that subcutaneously incubating NSC seeded scaffolds for 3 or 14 days could act as host specific preconditioning through exposure to FBR while retaining βIII tubulin expression of NSCs to further improve the SCI functional outcome observed with 28 day subcutaneous incubation.

Chitosan-based multifunctional oxygenating antibiotic hydrogel dressings for managing chronic infection in diabetic wounds

“…Oxygenated MACF dressings were prepared as described in our previous in vivo wound healing models 16,17,25 starting from purified chitosan (ChitoClear 43010, DDA ∼80% M w = 200 kDa, Primex, Siglufjörður, Iceland) to make MAC. 16,17,[26][27][28] To make MACF, MAC was fluorinated by dropwise addition of a methanol-PFOC mixture (Sigma Aldrich) solution in a 48 hour reaction. The polymer was then dialyzed against DI water, freeze dried, and stored in airtight containers until further use.…”

Recent progresses in neural tissue engineering using topographic scaffolds

“…It was demonstrated that the constructed 3D hydrogel scaffold could be used for repairing SCI by reducing inflammatory reaction, inhibiting glial scar formation and promoting axon regeneration. Hamrangsekachaee et al embedded a 3D hydrogel by subcutaneous preconditioning to improve nerve cells survive in an inflammatory environment for treatment of SCI (Figure 5B) [49]. Studying the mechanism of subcutaneous preconditioning by measuring neurogenic markers and several cytokines.…”

Mimicking the neural stem cell niche: An engineer’s view of cell: material interactions

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