Abstract
Purpose:
We previously reported that factors released from BD-TM4 cells, which are Müller cells stably expressing brain-derived neurotrophic factor (BDNF), enhanced the survival and neurite outgrowth of retinal ganglion cells (RGCs). Here, we examined the effectiveness of polyvinyl alcohol (PVA)-based hydrogels for encapsulation of BD-TM4 for in vivo applications. We also investigated the effects of crosslinkers and cell adhesion molecules within the hydrogels on the ability of cells to spread, proliferate, and secrete BDNF.
Methods:
PVA hydrogels, used for encapsulating BD-TM4 cells, were crosslinked with either a non-degradable linker, or a degradable linker by matrix metalloproteinases (MMP). PVA hydrogels were functionalized with the cell adhesion peptides (Arg-Gly-Asp (RGD) peptides). The ability of encapsulated BD-TM4 to spread and proliferate were studied with the LIVE/DEAD staining and the WST-8 assay. The BDNF levels released from the hydrogel capsules were quantified by ELISA. Neurite outgrowth of RGCs was evaluated by counting outgrowing axons after retinal explants subjected to optic nerve crush injury were cultured in encapsulated BD-TM4-conditioned medium for 6 days.
Results:
Regardless of the presence of RGD, BD-TM4 cells in hydrogels crosslinked with a non-degradable linker maintained spherical morphologies and released low-level BDNF at day 14. Cells in hydrogels crosslinked with a MMP degradable crosslinker with RGD exhibited spindle-like morphologies and extensive spreading, and released more than 10-fold more BDNF compared to those in other PVA hydrogels. RGD at concentration of 250 μM enhanced cell proliferation and the amount of BDNF released compared to RGD at 2.5, 25, and 2,500 μM. Encapsulated BD-TM4-conditioned medium increased neurite outgrowth in retinal explants.
Conclusions:
These results suggest that both the degradation of scaffolds via MMP and the presence of adhesion peptides are crucial in maintaining the BDNF-releasing function of BD-TM4 cells encapsulated in PVA hydrogels.
Keywords: 603 Muller cells •
618 nutritional factors •
615 neuroprotection