Supporting Information

3D Bioprinted Multiscale Composite Scaffolds Based on Gelatin Methacryloyl

(GelMA)/Chitosan Microspheres as a Modular Bioink for Enhancing 3D Neurite Outgrowth

and Elongation

Jiali Chena,b,c, Da Huanga,b,c, Ling Wange, Juedong Houa,b,c, Hongwu Zhanga,b,c, Yanbing Lia,b,c,

Shizhen Zhonga,b,c*, Yanfang Wanga,b,c, Yaobin Wua,b,c*, and Wenhua Huanga,b,c,d*

aDepartment of Human Anatomy, School of Basic Medical Sciences, Southern Medical University,

Guangzhou, 510515, P.R. China

bGuangdong Provincial Key Laboratory of Medical Biomechanics, School of Basic Medical

Sciences, Southern Medical University, Guangzhou, 510515, P.R. China

cGuangdong Engineering Research Center for Translation of Medical 3D Printing Application,

Guangzhou, 510515, P.R. China

dDepartment of Human Anatomy, School of Basic Medical Sciences, Guangdong Medical

University, Zhanjiang, 524002, P.R. China

eSchool of Biomedical Engineering, Southern Medical University, Guangzhou, 510515, P.R. China

To whom correspondence should be addressed. Tel.: +86-020-61648086. Fax: +86-020-61648524.

E-mail: huangwenhua2009@139.com, wuyaobin2018@smu.edu.cn, zhszh@fimmu.com.

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Figure S1. The 1H NMR spectra of GelMA. The peaks at 5.4 ppm and 5.6 ppm assigned to two H

methacrylic double bonds, and the peak around 1.87 ppm related to the methacrylate groups of

methacrylic acid.

Table S1. Monodisperse GC-MSs with different sizes.

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Inner phases

(μL/min)

Outer phases

(μL/min)

Flow rate ratio

(I:O)

Diameter

(μm)

MS-XL 15 75 1:5 350±21

MS-L 15 225 1:15 130±10

MS-S 15 300 1:20 112±8

MS-XS 15 450 1:30 89±9

MS-XXS 15 600 1:40 54±5

MS-M 20 300 1:15 126±9

MS-XXL 10 300 1:30 136±10

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Figure S2. The swelling ratio analysis of GC-MS immersed in the NGF solution. (a-c) The optical

microscopy images of GC-MS immersed in NGF solution. The diameters increased with the

increase of immersed time: (a) 0 min (b) 5 min (c) 20 min.(d) The statistical diagram of the

microsphere diameter, the X-axis is the time immersed NGF solution, the Y-axis is the diameter of

the microsphere.

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Figure S3. The confocal fluorescent microscopy images of NGF-Alexa Fluor 594 loaded on GC-MS

(a) and the release curve of GC-MS.

Figure S4. PC12 cells cultivation on TCP. The microscope fluorescent images of PC12 cells on TCP

staining with live/dead kit after cultured for 1 day (a) and 3 days (b), respectively.

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Table S2. The fluorescence intensity of PC12 cells proliferation on GC-MSs.

Day 1 Day 3 Day 5

control 1573416±257510 2844840±266446 3116954±623638

GC-MS 1506795±163481 2641219±77503 3330640±284679

GC-MS+NGF 1377797±126092 2646559±287892 3399494±134500

Figure S5. The SEM images of PC12 cells loaded GC-MS +NGF (a, b, c).

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Figure S6. Bioprinting of 3D GC-MS/GelMA multiscale composite scaffold. (a) The 3-layer 3D

GC-MS/GelMA structure was bioprinted. (b) The 4-layer 3D GC-MS/GelMA structure was

bioprinted. (c) The gross image of 3D GC-MS/GelMA structure.

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Figure S7. 3D views of confocal microscope fluorescent images of GC-MS/GelMA, GC-MS was

stained with FITC (green) and GelMA was stained with Nile red (red). Scale:200 μm. (a-c) The

intensity of pressure was 0.5 bar, the bioprinting speed was 5mm/s in the printing process. (d-f) The

intensity of pressure was 0.6 bar, the bioprinting speed was 5mm/s in the printing process.

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