Neural stem cells (NSCs) transplantation can reconstruct some neural circuits and functions, which is a promising treatment for spinal cord injury (SCI). However, the survival rate of NSCs directly transplanted to the lesion site is low, and the differentiation is easy to get out of control. 3D bioprinting can construct a cellular scaffold with a uniform distribution of NSCs and a precise and complex spinal-like structure, which is expected to provide a suitable microenvironment for NCSs and promote cell-scaffold and cell-cell interactions in the lesion area, which is of great significance for the neuronal regeneration and axon connection of extrinsic NSCs in vivo.
Recently, a team led by Professor Zhijun Zhang of the Suzhou Institute of Nanotechnology and Nanobionics at the Chinese Academy of Sciences has developed a 3D bioprinting strategy that can be used to create scaffolds for neural tissue carrying NSCs. The printed scaffolds can provide a good microenvironment for the growth and neural differentiation of NSCs, promote axon regeneration, reduce glial scar formation and optimize the formation of neural networks, and ultimately help restore the motor ability of SCI model rats and achieve SCI repair. The related results, "3D bioprinted neural tissue constructs for spinal cord injury repair," are published in Biomaterials.
Figure 1 Cross-linking reaction before and after HBC/HA/MA bio-ink 3D printing and 3D bioprinting diagram of neural tissue scaffolds containing NSCs
Bioink composition: hydroxypropyl chitosan (HBC), sulfhydrylated hyaluronic acid (HA-SH), vinyl sulfone hyaluronic acid (HA-VS) and matrigel (MA)
Process: HBC/ HA-sh /MA was mixed with HA-VS solution ice bath to obtain HBC/HA/MA-0.3 bio-ink (HBC 3% w/v, HA-SH0.3% w/v, HA-VS 0.3% w/v, HBC/HA/MA). MA 0.1% w/v) or BC/HA/MA-0.6 bio-ink (HBC 3% w/v, ha-SH0.6% w/v, ha-VS 0.6% w/v, MA 0.1% w/v). The HBC/HA/MA bio-ink was crosslinked to form HBC/HA/MA hydrogel by placing it at 37℃ for 2h. NSCs and HBC/HA/MA bioink mixed in ice bath for 5min can obtain cell-loaded bioink.
3D bioprinting conditions: extrusion bioprinting method; The syringe temperature is 10℃, the printing platform temperature is 37℃, and the printing pressure is about 20 kPa
1. Characterization of HBC/HA/MA bio-inks
1) Shear thinning characteristics, gel velocity and mechanical strength characterization
HBC/HA/MA bio-inks have typical shear dilution behavior. It has good fluidity at low temperatures, and can be quickly cross-linked into hydrogels after heating to 37 ° C. By comparing the gel time of HBC and HBC/HA/MA (the time when elastic modulus G '= loss modulus G "), it is found that the gel time of HBC and HBC/HA/MA bio-inks with the same concentration is similar, which indicates that HBC plays a major role in the rapid gelation of HBC/HA/MA bio-inks. In addition, the mechanical strength of HBC/HA/MA hydrogels is significantly higher than that of HBC and non-chemically bonded HBC/HA/MA hydrogels, and its mechanical strength is proportional to the HA concentration within a certain range (Figure 2).
2) Swelling rate, stability and morphological and structural characterization
Scanning electron microscopy (SEM) was used to observe the morphology and structure of HBC/HA/MA hydrogels, and it was found that the hydrogels had high pore structure with relatively uniform size (about 100 μm), and the pore size would be inversely proportional to the HA concentration within a certain range. The swelling rate and degradation behavior of HBC/HA/MA hydrogels were also observed by swelling ratio analysis and in vitro degradation tests. It was found that the swelling rate of HBC/HA/MA hydrogels was lower than that of HBC hydrogels, but the equilibrium water content related to cell culture was not significantly affected. In addition, hydrogels with a high degree of crosslinking have high stability and are not easily degraded (Figure 2).
Figure 2. Related characterization of HBC/HA/MA bio-inks
2. Preparation and related detection of 3D printed HBC/HA/MA scaffolds carrying NSCs
The authors prepared a square grid scaffold with HBC/HA/MA bio-ink containing NSCs (no deformation or collapse of the scaffold microstructure was observed during printing) and assessed the viability of NSCs in the scaffold with Live/Dead assay, which showed high cell viability and proliferation capacity. SEM results also showed that NSCs adhering to the scaffold fully extended after 7 days of culture, forming intercellular connections rich in filamentous pseudopods. They also investigated the differentiation ability of NSCs after biopprinting by immunofluorescence staining: they found that the cells in the scaffold expressed the astrocyte marker GFAP and the neuron-specific cytoskeleton protein Tuj1, and successfully differentiated into mature neurons. They also found that the concentration of HA in the scaffold had an important effect on the neuronal differentiation of NSCs, and NSCs in the HBC/HA/MA-0.3 scaffold had a high differentiation rate (Figure 3).
Figure 3 shows the relevant detection of HBC/HA/MA stents for NSCs
3. 3D printed linear array-like scaffolds loaded with NSCs were used for SCI rat treatment
1) Motor function assessment
The researchers printed NSCS-loaded linear array-like scaffolds (hereinafter referred to as "NSCs scaffolds") that simulated the natural white matter structure of the spinal cord, and then transplanted the scaffolds into the spinal cord transects of spinal cord injury model rats with T8-9 complete spinal cord transects. Finally, Basso-Beattie-Bresnahan (BBB) test and real-time monitoring by flexible pressure sensor were used to evaluate the muscle and joint movements of rats with NSCs scaffolds (therapeutic dose: 3×105), empty scaffolds and untreated groups. Compared with the control group, there was no obvious movement of hind limbs. The rats implanted with NSCs scaffolds showed significant recovery of motor function and simultaneous hip and knee ankle movements. The effect of SCI repair in the NSC-loaded stent transplantation group was better than that in the empty stent transplantation group and the control group (Figure 4A-E).
2) Histological analysis
In order to further study the therapeutic effect of NSCs scaffolds on SCI, the researchers conducted histological analysis of the injured spinal cord of mice in each group 12 weeks after implantation of scaffolds, detected the regeneration of neurons, axons and oligodendrocytes by immunofluorescence staining, and studied the growth of nerve fibers and myelination in the injured area of the spinal cord. It was found that Tuj1, NF, Oligo2, GAP-43, and myelin basic protein (MBP) were positively stained in the lesion area of the NSCs scaffold transplantation group, while GFPA expression was lower. This suggests that NSCs scaffolds promote axon and myelin formation, neuronal differentiation, and inhibit glial scar formation at the injured site (Figure 4F).
3) Survival and differentiation of NSCs after implantation
The investigators prepared GFP-NSCs scaffolds and implanted them into the lesion area. Samples were then taken 12 weeks after transplantation for immunofluorescence staining to monitor the fate of exogenous NSCs in vivo. Results showed that NSCs transplanted with scaffolds could survive for at least 12 weeks, and GFP was consistent with most Tuj1, NF, and Oligo2 localization, suggesting that surviving NSCs differentiated into TuJ1-positive neurons and oligo2-positive oligodendrocytes. The presence of neurons and oligodendrocytes that are not co-stained with GFP antibody suggests that endogenous NSCs may migrate to the injured area to participate in injury repair. These results all indicate that NSCS-loaded scaffolds provide a favorable microenvironment for NSCs (FIG. 4G-H).
Figure 4. Evaluation of spinal cord injury repair in SCI rats after implantation of NSCs scaffolds
Step 4 Summarize
HBC/HA/MA hydrogels have degradation rate, porosity and mechanical strength suitable for NSCs growth. NSCs scaffolds prepared by HBC/HA/MA hydrogels provide a good microenvironment for NSCs. NSCs in scaffolds have high survival rate, strong proliferation ability, and good neural differentiation ability. After transplantation, NSCs scaffolds can promote the formation of axons and myelin in the spinal cord injury area, inhibit the formation of glial scar, and significantly improve the motor function of SCI rats. The 3D bioprinted NSCs scaffold is expected to be used in neural tissue engineering and other regenerative medicine fields