*Article* **Injectable Hydrogel Guides Neurons Growth with Specific Directionality**

**Yun-Hsiu Tseng 1, Tien-Li Ma 1, Dun-Heng Tan 1, An-Jey A. Su 2,\*, Kia M. Washington 2, Chun-Chieh Wang 3, Yu-Ching Huang 4, Ming-Chung Wu 5,6,7,\* and Wei-Fang Su 1,4**

	- <sup>3</sup> National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan

**Abstract:** Visual disabilities affect more than 250 million people, with 43 million suffering from irreversible blindness. The eyes are an extension of the central nervous system which cannot regenerate. Neural tissue engineering is a potential method to cure the disease. Injectability is a desirable property for tissue engineering scaffolds which can eliminate some surgical procedures and reduce possible complications and health risks. We report the development of the anisotropic structured hydrogel scaffold created by a co-injection of cellulose nanofiber (CNF) solution and co-polypeptide solution. The positively charged poly (L-lysine)-r-poly(L-glutamic acid) with 20 mol% of glutamic acid (PLLGA) is crosslinked with negatively charged CNF while promoting cellular activity from the acid nerve stimulate. We found that CNF easily aligns under shear forces from injection and is able to form hydrogel with an ordered structure. Hydrogel is mechanically strong and able to support, guide, and stimulate neurite growth. The anisotropy of our hydrogel was quantitatively determined in situ by 2D optical microscopy and 3D X-ray tomography. The effects of PLLGA:CNF blend ratios on cell viability, neurite growth, and neuronal signaling are systematically investigated in this study. We determined the optimal blend composition for stimulating directional neurite growth yielded a 16% increase in length compared with control, reaching anisotropy of 30.30% at 10◦/57.58% at 30◦. Using measurements of calcium signaling in vitro, we found a 2.45-fold increase vs. control. Based on our results, we conclude this novel material and unique injection method has a high potential for application in neural tissue engineering.

**Keywords:** hydrogel; polypeptide; cellulose nanofiber; injectable; aligned structure; neuron; tissue engineering; three-dimensional tomography; calcium imaging
